Reprod Dom Anim 50, 474–483 (2015); doi: 10.1111/rda.12515 ISSN 0936–6768
Generation of Foxo3-targeted Mice by Injection of mRNAs Encoding Transcription Activator-like Effector Nucleases (TALENs) into Zygotes P Zhu1,*, Q Liu1,*, S Liu1, X Su1, W Feng1, X Lei1, J Liu1, K Cui1, B Huang1,2 and D Shi1 1 State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, Guangxi, China; 2Guangxi Experiment Centre of Science and Technology, Guangxi University, Nanning, Guangxi, China
Contents In this study, for exploring the mechanism of forkhead box O3 (Foxo3) participating in regulation of the activation of primordial oocytes, Foxo3-targeted mice were generated by injection of mRNAs encoding transcription activator-like effector nucleases (TALENs) into mouse zygotes. The TALEN sites were designed with high conservative homologous region among pig, bovine, buffalo and mouse by commercial biocompanies. The TALENs mutagenic non-homologous endjoining (NHEJ) repair activity were determined to be 31.3% in human embryonic kidney 293T (HEK-293T) cells by dual luciferase reporter assay system. Then, we firstly injected TALEN-mRNAs into the cytoplasm of mouse zygotes by micromanipulation, and four of 48 mouse blastocysts were identified as mutation by sequencing. Subsequently, by the method of TALEN-mRNAs injected into the zygotes with pronucleus micromanipulation technique, we obtained seven Foxo3 mutants of 20 FVB/NJ backgrounds mice which were Foxo3-independent alleles with frameshift and deletion mutations. It was very interesting that all seven were heterozygous mutants (Foxo3/+), and the gene mutagenesis rates of the mice reached 35%. The five Foxo3 mutant females were all infertile in the following 6 months after birth. The histological examination results showed that there were rare primordial follicles and primary follicles in the ovary of Foxo3 mutant compared to that of wide-type female mice. Moreover, one of two mutant males was subfertile and another was fertile normally. Those results suggested that the mutant of Foxo3 severely affected the fertile ability of female and perhaps male in some degree; furthermore, an even more efficient TALENsbased gene mutation method has been established to be poised to revolutionize the study of mouse and other species genetics.
Introduction The ability to precisely modify the mouse genome experimentally has had a considerable impact over the last 25 years in diverse areas of biomedical research and has made the mouse one of the most important model organisms in the laboratory today. Alterations in the genome are conventionally made by the process of gene targeting in embryonic stem cell via homologous recombination (Doyle et al. 2012). Using homologous recombination, whole genes or exons can be deleted from the mouse genome and the phenotypic consequences of these knockout models can deliver important information concerning gene function. However, the
*Those authors contributed to this article equivalently.
generation of knockout animals via the traditional method, such as homologous recombination, is a timeconsuming and laborious process, and therefore, a more-efficient tool is preferred for generating knockout mouse. Targeted gene knockdown by RNAi has provided researchers with a rapid, inexpensive and highthroughput alternative to homologous recombination (McManus and Sharp 2002). Yet, knockdown by RNAi is incomplete, varies between experiments and laboratories, has unpredictable off-target effects and provides temporary inhibition of gene function or can be permanently knockdown using transgenic approach in vivo or lentiviral vectors in vitro. In the past decade, a new approach has emerged that enables investigators to manipulate virtually any gene in a diverse range of cell types and organisms. The versatility of this approach is facilitated by the programmability of the DNA-binding domains that are derived from zinc-finger and transcription activator-like effector (TALE) proteins. This combination of simplicity and flexibility has catapulted zinc-finger nucleases (ZFNs) (Urnov et al. 2010; Carroll 2011) and transcription activator-like effector nucleases (TALENs)( Ikmi et al. 2014; Moghaddassi et al. 2014; Sun et al. 2014). Also, in the past years, a new approach has been developed and applied, it is just the clustered regulatory interspaced short palindromic repeats CRISPR/Cas (CRISPR-associated)-based RNA-guided DNA endonucleases (Cho et al. 2013; Cong et al. 2013; Mali et al. 2013). Up to now, these engineered TAL effector nucleases (TALENs) have been successfully applied to disrupt gene function in the yeast (Li et al. 2011), rice (Li et al. 2014), zebrasfish (Tatsumi et al. 2014), bovine (Yang et al. 2014), mouse (Nakagawa et al. 2014), human (Fadel et al. 2014) and so on. Knocking-out genes contain both protein coding genes (Bradley et al. 2012) and microRNA (Prosser et al. 2011; Zhang et al. 2013). Foxo3 is a member of the FOXO (forkhead box class O) transcription factors family and consists of four exons and produce a protein of a 673 amino acids. It has roles in cell cycle arrest (Medema et al. 2000; Alvarez et al. 2001), apoptosis (Dijkers et al. 2000; Modur et al. 2002) and DNA repair (Tran et al. 2002; Luo et al. 2006) among other functions. Consistent with their ability to block cell growth, inactivation of FOXO proteins is thought to be a critical event in oncogenic © 2015 Blackwell Verlag GmbH
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transformation (Greer and Brunet 2005; Fu and Tindall 2008). Deletion of all FOXO1, Foxo3 and FOXO4 alleles in adult mice induced a cancer prone phenotype, supporting a tumour-suppressing function of these proteins (Paik et al. 2007). In addition to their role in cell cycle regulation, FOXO transcription factors also regulate glucose metabolism in various organs (Matsumoto et al. 2007; Haeusler et al. 2010) and increase the resistance to oxidative stress (Kops et al. 2002; Brunet et al. 2004). Furthermore, the FOXO orthologue DAF-16 controls lifespan extension in Caenorhabditis elegans together with homologues of the human insulin receptor and the phosphatidylinositol 3-kinase (DAF-2 and AGE-1) (Lin et al. 1997; Ogg et al. 1997). Specifically Foxo3/ female mice had a very singular phenotype which they were initially fertile and subsequently infertile after 15 weeks of age (Castrillon et al. 2003; Hosaka et al. 2004). The activation of primordial follicle was presented in Foxo3 knockdown pigs (Moniruzzaman et al. 2010), but the mechanism remains to be further clarified. In this study, we were keen to produce Foxo3 knockdown mice by TALEN-based targeting technique and, consequently, able to establish an efficient genetargeting system for creating heritable mutations to apply for the study of mouse and other species genetics.
Materials and methods TALENs and reporters Plasmids encoding the TALENs pCS2-peas-T and reporter pSSA-LUC used in this study were obtained from viewsolid biotech (Beijing, China). Reporter pRGS were prepared as previously described (Kim et al. 2011). Briefly, oligonucleotides that contained target sites were synthesized (Sangon biotech, Shanghai, China) and annealed in vitro. The annealed oligonucleotides were ligated into the pRGS and pSSA-LUC (Table 1). After DNA for each of the Foxo3-TALEN and reporter plasmids were prepared using the Qiagen Endo-free Maxi-prep kit as directed by the manufacturer, reporter plasmid co-transfection of the Foxo3-TALEN vectors into HEK293T cells led to nuclease-dependent gene repair and enhanced green fluorescent protein (eGFP) and luciferase were expressed.
Cell culture and transfection HEK293T cells were maintained in Dulbecco’s modified Eagle medium (DMEM, Invitrogen) supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin, and 10% foetal bovine serum. Cells were transfected using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) at a weight ratio of 1 : 1 : 2 (plasmid encoding a TALEN: plasmid encoding the other TALEN: pRGS reporter) or 100 ng plasmid encoding a TALEN: 100 ng plasmid encoding the other TALEN: 50 ng pSSA-LUC reporter: 10 ng pRL-TK (Promega, Beijing, China). Dual luciferase reporter assay The 293T cells were seeded in 24-well plates. After 24-h incubation, cells were transfected with TALENs and pSSA-Luc (Sajwan et al. 2013; Aryan et al. 2013). Fortyeight hours after transfection, the cells were assayed by both firefly and renilla luciferase using the dual luciferase assay system (Promega) according to manufacturer’s instructions. All transfection experiments were conducted in triplicate and repeated three times independently. Preparation and cytoplasm or pronuclear microinjection of TALEN mRNA The mRNAs were generated with T7 RNA polymerase from 1 lg of linearized pCS2-peas-T construct using the mMessage mMachine T7 Kit (Life Technologies, 2013, Guangzhou, China) and purified using LiCl according to the manufacturer’s instructions. Purified mRNAs were diluted to 50 ng/ll for cytoplasm microinjection) or 100 ng/ll for pronuclear microinjection in 1 mM Tris.HCl pH 7.5/0.1 mM EDTA and were microinjected into the cytoplasm of fertilized oocytes. A pair of primers including Ftln-F and Ftln-R were designed and used to amplify the region containing the Foxo3 target site of the injected zygotes (Table 1). The PCR products were sequenced in BGI (Shenzhen, China), and gene mutation was analysed. Generation of Foxo3-targeted mice and genotyped Foxo3 knockout mice were generated by pronucleus microinjection TALEN-mRNAs in Cyagen (Guangzhou, China). Genomic DNA was isolated from tail tips of
Table 1. Primers used in this study Products
Amplicon length(bp)
Frgs
/
Fluc
/
Ftln
653
Primer name
Sequences(50 ?30 )
Frgs-F Frgs-R Fluc-F Fluc-R Ftln-F Ftln-R
aattcCCTGTACGTGGCCCCTGCAGAGGCCGGAGCTGCAGGCGAGCCCGGCCAAGCCCTg gatccAGGGCTTGGCCGGGCTCGCCTGCAGCTCCGGCCTCTGCAGGGGCCACGTACAGGg gatcCCTGTACGTGGCCCCTGCAGAGGCCGGAGCTGCAGGCGAGCCCGGCCAAGCCCTg tcgaAGGGCTTGGCCGGGCTCGCCTGCAGCTCCGGCCTCTGCAGGGGCCACGTACAGGg CGGACTAGGCTCCAACAC ACCTGTCCTATGCCGACC
The annealed oligonucleotides Frgs and Fluc were ligated into the pRGS and pSSA-LUC, respectively. Primers Ftln was used to amplify the region containing the Foxo3 target site.
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founder mice. The primers Ftln-F and Ftln-R were used to amplify the region containing the Foxo3 target site from wild-type individuals or founders. The PCR products that included TALEN-target sites were purified using the Gel Extraction Kit (Tiangen, Beijing, China) and cloned into the pEASY-T-Blunt vector using the pEASYT-Blunt PCR Cloning Kit (Transgen, Beijing, China). Cloned plasmids were sequenced in BGI (Shenzhen, China), and gene mutation was analysed.
Histology Ovaries were fixed for 3–5 h in 2.5% glutaraldehyde and 2.5% paraformaldehyde in 0.083 M sodium cacodylate buffer, pH 7.2, 4°C. They were then washed in 0.1 M sodium cacodylate buffer, pH 7.2, for 24 h before embedding in JB-4 (glycol methacrylate) plastic (Polysciences, Inc., Warrington, PA, USA). Sections, 2 lm thick, were stained with haematoxylin and eosin.
(a)
(b)
(c)
Fig. 1. FOXO3 genomic structure, TALEN binding sites and report plasmids structure. (a) Genomic structure of the mouse FOXO3 (upper panel) with an enlargement of exon 2 (lower panel), showing the binding sites of for TALEN together with the binding sites of the PCR primers, Ftln-F and Ftln-R used to genotype the mutant alleles. The FOXO3 TALEN target sequence, with Left and Right TALEN monomer binding sites underlined were shown. The TALEN sites were high conservative homologous region among pig, bovine, buffalo and mouse. (b) Outline of the nuclease reporter pRGS assay. TALEN-FOXO3 targeted sequences were cloned into the locations between the report gene RFP and eGFP, Reporter plasmid co-transfection of the TALEN-FOXO3 vectors into HEK293T cells led to nuclease dependent gene repair and eGFP was expressed, as shown by green fluorescence. The red fluorescence from RFP was used as an indicator of plasmid transfection efficiency. (c) Outline of the nuclease reporter pSSA-LUC assay. TALEN-FOXO3 targeted sequences were cloned into the locations between the luciferase gene segments. Reporter plasmid co-transfection of the TALEN-FOXO3 vectors and pRL-TK into HEK293T cells led to nuclease dependent gene repair and luciferase was expressed. The renilla fluorescence was used as an indicator of plasmid transfection efficiency
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482 factors. Proc Natl Acad Sci 97, 1495– 1500. Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U, 2009: Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509–1512. Bradley A, Anastassiadis K, Ayadi A, Battey JF, Bell C, Birling M-C, Bottomley J, Brown SD, B€ urger A, Bult CJ, 2012: The mammalian gene function resource: the international knockout mouse consortium. Mamm Genome 23, 580–586. Brinster RL, Chen HY, Trumbauer ME, Yagle MK, Palmiter RD, 1985: Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc Natl Acad Sci 82, 4438–4442. Brunet A, Sweeney LB, Sturgill JF, Chua KF, Greer PL, Lin Y, Tran H, Ross SE, Mostoslavsky R, Cohen HY, 2004: Stressdependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science 303, 2011–2015. Cade L, Reyon D, Hwang WY, Tsai SQ, Patel S, Khayter C, Joung JK, Sander JD, Peterson RT, Yeh J-RJ, 2012: Highly efficient generation of heritable zebrafish gene mutations using homo-and heterodimeric TALENs. Nucleic Acids Res 40, 8001–8010. Carlson DF, Tan W, Lillico SG, Stverakova D, Proudfoot C, Christian M, Voytas DF, Long CR, Whitelaw CBA, Fahrenkrug SC, 2012: Efficient TALEN-mediated gene knockout in livestock. Proc Natl Acad Sci 109, 17382–17387. Carroll D, 2011: Genome engineering with zinc-finger nucleases. Genetics 188, 773–782. Castrillon DH, Miao L, Kollipara R, Horner JW, DePinho RA, 2003: Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a. Science 301, 215–218. Chen S, Oikonomou G, Chiu CN, Niles BJ, Liu J, Lee DA, Antoshechkin I, Prober DA, 2013: A large-scale in vivo analysis reveals that TALENs are significantly more mutagenic than ZFNs generated using context-dependent assembly. Nucleic Acids Res 41, 2769–2778. Cho SW, Kim S, Kim JM, Kim J-S, 2013: Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31, 230–232. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, 2013: Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823. Dijkers PF, Lammers J-WJ, Koenderman L, Coffer PJ, 2000: Expression of the proapoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1. Curr Biol 10, 1201– 1204. Doyle A, McGarry MP, Lee NA, Lee JJ, 2012: The construction of transgenic and gene knockout/knockinmousemodels ofhuman disease. Transgenic Res 21, 327–349. Fadel HJ, Morrison JH, Saenz DT, Fuchs JR, Kvaratskhelia M, Ekker SC, Poeschla
P Zhu, Q Liu, S Liu, X Su, W Feng, X Lei, J Liu, K Cui, B Huang and D Shi EM, 2014: TALEN knockout of the PSIP1 gene in human cells: analyses of HIV-1 replication and allosteric integrase inhibitor mechanism. J Virol 88, 9704– 9717. Fu Z, Tindall D, 2008: FOXOs, cancer and regulation of apoptosis. Oncogene 27, 2312–2319. Geurts AM, Cost GJ, Freyvert Y, Zeitler B, Miller JC, Choi VM, Jenkins SS, Wood A, Cui X, Meng X, 2009: Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325, 433–433. Greer EL, Brunet A, 2005: FOXO transcription factors at the interface between longevity and tumor suppression. Oncogene 24, 7410–7425. Haeusler RA, Kaestner KH, Accili D, 2010: FoxOs function synergistically to promote glucose production. J Biol Chem 285, 35245–35248. Hosaka T, Biggs WH, Tieu D, Boyer AD, Varki NM, Cavenee WK, Arden KC, 2004: Disruption of forkhead transcription factor (FOXO) family members in mice reveals their functional diversification. Proc Natl Acad Sci USA 101, 2975– 2980. Ikmi A, McKinney SA, Delventhal KM, Gibson MC, 2014: TALEN and CRISPR/ Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis. Nat Commun 5, 5486. Kim H, Um E, Cho S-R, Jung C, Kim H, Kim J-S, 2011: Surrogate reporters for enrichment of cells with nucleaseinduced mutations. Nat Methods 8, 941– 943. Kops GJ, Dansen TB, Polderman PE, Saarloos I, Wirtz KW, Coffer PJ, Huang T-T, Bos JL, Medema RH, Burgering BM, 2002: Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 419, 316–321. Li T, Huang S, Jiang WZ, Wright D, Spalding MH, Weeks DP, Yang B, 2011: TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res 39, 359–372. Li T, Liu B, Chen CY, Yang B, 2014: TALEN utilization in rice genome modifications. Methods 69, 9–16. Lin K, Dorman JB, Rodan A, Kenyon C, 1997: daf-16: an HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278, 1319–1322. Liu J, Li C, Yu Z, Huang P, Wu H, Wei C, Zhu N, Shen Y, Chen Y, Zhang B, 2012: Efficient and specific modifications of the drosophila genome by means of an easy TALEN strategy. J Genet Genomics 39, 209–215. Liu Y, Lv X, Tan R, Liu T, Chen T, Li M, Liu Y, Nie F, Wang X, Zhou P, 2014: A modified TALEN-based strategy for rapidly and efficiently generating knockout mice for kidney development studies. PLoS One 9, e84893. Luo X, Puig O, Hyun J, Bohmann D, Jasper H, 2006: Foxo and Fos regulate the
decision between cell death and survival in response to UV irradiation. EMBO J 26, 380–390. Ma S, Zhang S, Wang F, Liu Y, Liu Y, Xu H, Liu C, Lin Y, Zhao P, Xia Q, 2012: Highly efficient and specific genome editing in silkworm using custom TALENs. PLoS One 7, e45035. Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, Eichtinger M, Jiang T, Foley JE, Winfrey RJ, Townsend JA, 2008: Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell 31, 294–301. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM, 2013: RNA-guided human genome engineering via Cas9. Science 339, 823–826. Matsumoto M, Pocai A, Rossetti L, DePinho RA, Accili D, 2007: Impaired regulation of hepatic glucose production in mice lacking the forkhead transcription factor Foxo1 in liver. Cell Metab 6, 208–216. McManus MT, Sharp PA, 2002: Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 3, 737–747. Medema RH, Kops GJ, Bos JL, Burgering BM, 2000: AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature 404, 782–787. Modur V, Nagarajan R, Evers BM, Milbrandt J, 2002: FOXO proteins regulate tumor necrosis factor-related apoptosis inducing ligand expression Implications for PTEN mutation in prostate cancer. J Biol Chem 277, 47928–47937. Moghaddassi S, Eyestone W, Bishop CE, 2014: TALEN-mediated modification of the bovine genome for large-scale production of human serum albumin. PLoS One 9, e89631. Moniruzzaman M, Lee J, Zengyo M, Miyano T, 2010: Knockdown of FOXO3 induces primordial oocyte activation in pigs. Reproduction 139, 337–348. Moscou MJ, Bogdanove AJ, 2009: A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501. Nakagawa Y, Yamamoto T, Suzuki K, Araki K, Takeda N, Ohmuraya M, Sakuma T, 2014: Screening methods to identify TALEN-mediated knockout mice. Exp Anim 63, 79–84. Ogg S, Paradis S, Gottlieb S, Patterson GI, Lee L, Tissenbaum HA, Ruvkun G, 1997: The Fork head transcription factor DAF16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389, 994–999. Paik J-H, Kollipara R, Chu G, Ji H, Xiao Y, Ding Z, Miao L, Tothova Z, Horner JW, Carrasco DR, 2007: FoxOs are lineagerestricted redundant tumor suppressors and regulate endothelial cell homeostasis. Cell 128, 309–323. Panda SK, Wefers B, Ortiz O, Floss T, Schmid B, Haass C, Wurst W, K€ uhn R, 2013: Highly efficient targeted mutagenesis in mice using TALENs. Genetics 195, 703–713.
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to measure activity in mammalian cells HEK293T, repair activity was analysed using SPSS version 19.0 (SPSS, Inc., Chicago, IL, USA), and a p-value of less than 0.05 was considered to be significant.
Results Selection of TALEN-target site and activity assay in vitro For exploring the possibility of target same site of Foxo3 gene with different species, TALEN sites were designed for Foxo3 which were homologous among pig, bovine, buffalo and mouse (Fig. 1a). TALEN-Foxo3 construct was cloned into a mammalian expression vector and functionally validated using pRGS reporter assay or pSSA-LUC reporter assay in HEK293T cells. Firstly, we used pRGS reporter to investigate whether this TALEN can cleave the target sequence incorporated between the RFP and GFP sequences in HEK293T cells (Fig. 1b). In this reporter, the gfp sequence was fused to the end of rfp sequence and inactivated by frameshift mutation and only expressed when the target sequence was cleaved by site-specific nucleases, which caused some small, frameshifting
insertions or deletions around the target sequence by means of error-prone non-homologous end-joining repair of the double-strand breaks. In this study, Foxo3-TALENs can recognize and cleave the target DNA sequence (Fig. 2a–c) demonstrated by GFP-expressing cells only obtained from the treatment of co-transfection with the in vitrotranscribed Foxo3-TALENs. Then, we used a pSSLUC reporter to measure their activity in mammalian cells. Site-specific nucleases were fused to the luciferase sequence (Fig. 1c). Luciferase is expressed only when the target sequence is cleaved by site-specific nucleases. By co-transfecting TALENS, pSS-LUC and renilla, we found that the TALENS mutagenic nonhomologous end-joining repair efficiency reached 31.3% (Fig. 2d). Generation and identification of Foxo3 mutant mice Firstly, in vitro-transcribed Foxo3-TALENs mRNAs were injected into the cytoplasm of fifty-five mouse zygotes by micromanipulation, and subsequently, those injected zygotes were in vitro cultured into blastocyst
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Fig. 3. PCR and genotypic analysis revealed mutations in 4 mouse blastocyst injected with FOXO3 TALENs. (a) Blastocyst 52 and 30 were heterozygous, 46 and 53 were deleted 52 bp and 88 bp. (b) No mutations in others blastocyst
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Fig. 4. Created FOXO3 knockout mice by TALEN-mediated gene targeting and sequence information of FOXO3 mutant alleles. (a) Seven maturation Founders by TALEN-mediated gene targeting. (b) Mutations of 20 pups by PCR detecting. (c) Sequences of FOXO3 mutations in seven F0 mice lines. Deletions are indicated by dashes with gray highlight and insertions are highlighted in gray. The DNA sequences to which the TALEN monomers were designed are highlighted in red. (d) DNA sequencing result of seven F0 mice lines. (e) To determine whether the FOXO3 mutations were heritable, we screened all the founders and found that the two male mice transmitted target site mutations to their F1 mice, demonstrating successful germline transmission. 1–10, F1 female mice of NO.5 F0 male, 11–19, F1 male mice of NO.5 F0 male, 20, F1 mouse of NO.4 F0 male, 21, NO.4 F0 male, 22, NO.5 F0 male
(d)
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stage. The following PCR and genotypic analysis results indicated that there were four mutants among 48 analysed embryos (Fig. 3a,b). © 2015 Blackwell Verlag GmbH
Secondly, pronuclear microinjections of TALENFoxo3 mRNAs (50 ng/ll) were conducted. Then, the pups were screened for the presence of mutant alleles by
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sequence analysis of PCR products covering the targeted exon. Seven of 20 mice (35%) were identified as Foxo3 heterozygous mutant (Foxo3/+) (Fig. 4, Table 2).Thirdly, sequence alignment results showed that the Foxo3 sequences of two male mice were deleted 34 or 51 bp, respectively. Those of the other female were added 2 bp, or deleted 15, 47 or 100 bp, respectively. Phenotypes observation of Foxo3 mutant mice To determine whether the Foxo3 mutations were heritable, two mutant female mice were caged with one mutant male for 2 weeks. And the other mutant female was caged with a wide-type male for 2 weeks. Unfortunately, none mutant female was pregnant. Therefore, we arranged the two mutant males (Nos. 4 and 5) to cage with two wide-type female, respectively; subsequently, the two female all got pregnancy. No.5 mutant male produced normal number of pups compared to that of wild type; however, No.4 mutant male only got six pups (Table 3) during a long caged period. The ovary of mutant (Foxo3+/) female mice was collected, and no corpus luteum was observed, which maybe suggest Foxo3 knockdown would disrupt ovulation. The histological examination results showed there were rare primordial and early follicles were observed compared to that of wide-type female (Fig. 5).
Discussion With the development of new and affordable methods for whole-genome sequencing, scientists are poised to deliver upon the promises of the genomic revolution to transform basic science and personalized medicine. However, the use of homologous recombination has been hampered by several factors, including the low efficiency at which engineered constructs are correctly inserted into the chromosomal target site, the need for time-consuming and labour-insensitive selection/screening strategies, and the potential for adverse mutagenic effects. While current RNAi technology has enabled to increase the efficiency of homologous recombination, it is still limited by variation in the degree and timing of knockdown, and off-target effects. In contrast, the
Table 3. The reproductive ability of NO.4 male mouse Female and age 1 2 3 4 5 6
(1.5 (1.5 (1.5 (1.5 (1.5 (1.5
month) month) month) month) month) month)
Age of NO.4 male
Caged time
Born
1.5 month 1.5 month 1.5 month 3 month 3 month 3 month
Caged for 1.5 month
3 3 0 0 0 0
Caged for 2 month
No.4 mutant male got six pups which suggested it maybe subfertile.
ability to directly and specifically disrupt a gene of interest offers the possibility to perform intricate reverse genetic experiments on any gene, in any organism. In the past decade, zinc-finger nucleases (Beerli et al. 1998, 2000) and transcription activator-like effector nucleases (Boch et al. 2009; Moscou and Bogdanove 2009) had been developed and applicated. They had been successfully applied to disrupt gene function in different organism with high editing rates. A major drawback of ZFNs, however, is the elaborate and time-consuming experimental selection process (Maeder et al. 2008). Although simplified methods, such as modular assembly and CoDA, have been reported (Sander et al. 2011), the quality of ZFNs generated by such platforms is controversial (Ramirez et al. 2008) or not determined yet. In zebrafish, TALENs are proved significantly more likely to be mutagenic and induce an average of 10-fold more mutations than ZFNs, 20% and 2% for TALENs and ZFNs, respectively (Chen et al. 2013). Up to date, in the family of genomic editing toolbox, TALEN has shown to be an efficient, rapid, specific and economic method with a wide range of applications. TALEN-induced indels are often variable in length, often leading to a frameshift. It is worth noting that different pairs of TALENs yield indels with different efficiencies, ranging from 0% to approximately 65% in flies and 0% to approximately 50% in zebrafish (Wei et al. 2013). In other laboratories, seven TALENs targets in various endogenous zebrafish genes induced targeted indel mutations with high efficiencies ranging from 2% to 76% (Cade et al. 2012). DF Carlson found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in
Table 2. The presence of mutant alleles of 7 mice Mouse
Gender
4 5 6 8 12 17 18
Male Male Female Female Female Female Female
Mutations Delete 47 bp Delete 51 bp Added 2 bp Delete 15 bp Delete 34 bp Delete 100 bp Not detected by plasmids sequencing picks by PCR products sequencing
Change protein Out of frame Delete 17 aa Out of frame Delete 5 aa Out of frame Out of frame but was mixed
Phenotypes Subfertile Normal reproduction Infertile Infertile Infertile Infertile Infertile
The presence of mutant alleles by sequence analysis of PCR products covering the targeted exon, and seven of these mice (35%) were identified as founders harboring mutant Foxo3 alleles.
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(a)
(b)
Fig. 5. Histological analysis of Foxo3a–/+ and control ovaries at 20 weeks. (a) Histology of widetype mouse ovary. (b) Histology of number 12 female founder ovary. Green arrows: small follicles
primary cells. Cytoplasmic injections of TALENmRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analysed (Carlson et al. 2012). In our experiment, the success rate we observed is 35% (>20%), similar to those described in many other organisms (Liu et al. 2012; Ma et al. 2012; Panda et al. 2013). And the TALEN-based editing was associated with deletions in size 47, 51 and 100 bp, respectively, which are longer than others report of small detections ranging in size from 1 to 7 bp (Liu et al. 2012; Ma et al. 2012; Watanabe et al. 2012). In addition, large deletions and inversions of sequences that are longer than 6 kb have been obtained in pigs by targeting two TALEN pairs to the same chromosome (Carlson et al. 2012). In our study, mutations in blastocysts generated by cytoplasm microinjections (8.3%) were lower than that of generated by pronucleus microinjection (35%). However, Liu et al. (2014) demonstrate that cytoplasm microinjection may result in a higher efficiency (85.7%) than that of pronucleus microinjection (30.4%). In 1985, Brinster (Brinster et al. 1985) observed that the integration frequency of linearized DNA injected in the cytoplasm or male pronucleus of mouse zygotes was 3.4% or 25–30%, respectively. Geurts (Geurts et al. 2009) revealed that rate of successful mutagenesis of lgM gene by pronucleus or cytoplasm microinjection ZFN mRNAs into the zygotes was 13–16% or 2–13% in rat, respectively. The reasons for different abilities of DNA or mRNA function after cytoplasm or pronucleus injection maybe caused by strain, gene or proficiency in injection. Foxo3/ female mice have a very singular phenotype which are initially fertile and subsequently infertile after 15 weeks of age (Castrillon et al. 2003; Hosaka et al. 2004). Foxo3 knockdown induced primordial oocyte activation in pigs (Moniruzzaman et al. 2010). In our study, all Foxo3 gene knockout females were infertile,
References Alvarez B, Martınez-A C, Burgering BM, Carrera AC, 2001: Forkhead transcription factors contribute to execution of the mitotic programme in mammals. Nature 413, 744–747.
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and their primordial oocytes were rare. The two mutant males, one of them in which the Foxo3 sequence was deleted 51 bp was normal fertile; however, another was subfertile in which Foxo3 sequence was deleted 34 bp. It maybe indicate that Foxo3 influence male reproduction, but more researches should be conducted to determine this point.. In conclusion, our study establishes that the TALENmediated targeting is an efficient method for creating heritable mutations in Foxo3 exon2 locus of the mouse genome, and also, this TALEN sites are homologous to bovine and sus, the work using these TALENs to edit bovine or sus genome is being in progress (data not shown). Taken together, these data suggest that TALEN-mediated in vivo mutagenesis might expedite the creation of genetically engineered mouse models or livestock and thereby help to accelerate functional genomic research. Acknowledgements This work was funded by the China Transgenic Project (2011ZX08007-003) and the National Natural Science Fund (Grant No. 31160457 and 31401267), the Scientific Research Foundation of Guangxi University (Grant No. XGZ130880), Guangxi Natural Science Foundation (Grant No. 2014GXNSFCB118003), and Guangxi Experiment Centre of Science and Technology (YXKT2014005).
Conflict of interest None of the authors have any conflict of interest to declare.
Author contributions Peng Zhu and Qingyou Liu designed and conducted the experiment, Shuai Liu, Xiaoping Su, Wanyou Feng, Xiaocan Lei, Kuiqing Cui and Jinfeng Liu conducted part of experiments, Ben Huang and Deshun Shi analysed data and drafted manuscript.
Aryan A, Anderson MA, Myles KM, Adelman ZN, 2013: TALEN-based gene disruption in the dengue vector Aedes aegypti. PLoS One 8, e60082. Beerli RR, Segal DJ, Dreier B, Barbas CF, 1998: Toward controlling gene expression at will: specific regulation of the erbB-2/
HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. Proc Natl Acad Sci 95, 14628–14633. Beerli RR, Dreier B, Barbas CF, 2000: Positive and negative regulation of endogenous genes by designed transcription
482 factors. Proc Natl Acad Sci 97, 1495– 1500. Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U, 2009: Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509–1512. Bradley A, Anastassiadis K, Ayadi A, Battey JF, Bell C, Birling M-C, Bottomley J, Brown SD, B€ urger A, Bult CJ, 2012: The mammalian gene function resource: the international knockout mouse consortium. Mamm Genome 23, 580–586. Brinster RL, Chen HY, Trumbauer ME, Yagle MK, Palmiter RD, 1985: Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc Natl Acad Sci 82, 4438–4442. Brunet A, Sweeney LB, Sturgill JF, Chua KF, Greer PL, Lin Y, Tran H, Ross SE, Mostoslavsky R, Cohen HY, 2004: Stressdependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science 303, 2011–2015. Cade L, Reyon D, Hwang WY, Tsai SQ, Patel S, Khayter C, Joung JK, Sander JD, Peterson RT, Yeh J-RJ, 2012: Highly efficient generation of heritable zebrafish gene mutations using homo-and heterodimeric TALENs. Nucleic Acids Res 40, 8001–8010. Carlson DF, Tan W, Lillico SG, Stverakova D, Proudfoot C, Christian M, Voytas DF, Long CR, Whitelaw CBA, Fahrenkrug SC, 2012: Efficient TALEN-mediated gene knockout in livestock. Proc Natl Acad Sci 109, 17382–17387. Carroll D, 2011: Genome engineering with zinc-finger nucleases. Genetics 188, 773–782. Castrillon DH, Miao L, Kollipara R, Horner JW, DePinho RA, 2003: Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a. Science 301, 215–218. Chen S, Oikonomou G, Chiu CN, Niles BJ, Liu J, Lee DA, Antoshechkin I, Prober DA, 2013: A large-scale in vivo analysis reveals that TALENs are significantly more mutagenic than ZFNs generated using context-dependent assembly. Nucleic Acids Res 41, 2769–2778. Cho SW, Kim S, Kim JM, Kim J-S, 2013: Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31, 230–232. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, 2013: Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823. Dijkers PF, Lammers J-WJ, Koenderman L, Coffer PJ, 2000: Expression of the proapoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1. Curr Biol 10, 1201– 1204. Doyle A, McGarry MP, Lee NA, Lee JJ, 2012: The construction of transgenic and gene knockout/knockinmousemodels ofhuman disease. Transgenic Res 21, 327–349. Fadel HJ, Morrison JH, Saenz DT, Fuchs JR, Kvaratskhelia M, Ekker SC, Poeschla
P Zhu, Q Liu, S Liu, X Su, W Feng, X Lei, J Liu, K Cui, B Huang and D Shi EM, 2014: TALEN knockout of the PSIP1 gene in human cells: analyses of HIV-1 replication and allosteric integrase inhibitor mechanism. J Virol 88, 9704– 9717. Fu Z, Tindall D, 2008: FOXOs, cancer and regulation of apoptosis. Oncogene 27, 2312–2319. Geurts AM, Cost GJ, Freyvert Y, Zeitler B, Miller JC, Choi VM, Jenkins SS, Wood A, Cui X, Meng X, 2009: Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325, 433–433. Greer EL, Brunet A, 2005: FOXO transcription factors at the interface between longevity and tumor suppression. Oncogene 24, 7410–7425. Haeusler RA, Kaestner KH, Accili D, 2010: FoxOs function synergistically to promote glucose production. J Biol Chem 285, 35245–35248. Hosaka T, Biggs WH, Tieu D, Boyer AD, Varki NM, Cavenee WK, Arden KC, 2004: Disruption of forkhead transcription factor (FOXO) family members in mice reveals their functional diversification. Proc Natl Acad Sci USA 101, 2975– 2980. Ikmi A, McKinney SA, Delventhal KM, Gibson MC, 2014: TALEN and CRISPR/ Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis. Nat Commun 5, 5486. Kim H, Um E, Cho S-R, Jung C, Kim H, Kim J-S, 2011: Surrogate reporters for enrichment of cells with nucleaseinduced mutations. Nat Methods 8, 941– 943. Kops GJ, Dansen TB, Polderman PE, Saarloos I, Wirtz KW, Coffer PJ, Huang T-T, Bos JL, Medema RH, Burgering BM, 2002: Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 419, 316–321. Li T, Huang S, Jiang WZ, Wright D, Spalding MH, Weeks DP, Yang B, 2011: TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res 39, 359–372. Li T, Liu B, Chen CY, Yang B, 2014: TALEN utilization in rice genome modifications. Methods 69, 9–16. Lin K, Dorman JB, Rodan A, Kenyon C, 1997: daf-16: an HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278, 1319–1322. Liu J, Li C, Yu Z, Huang P, Wu H, Wei C, Zhu N, Shen Y, Chen Y, Zhang B, 2012: Efficient and specific modifications of the drosophila genome by means of an easy TALEN strategy. J Genet Genomics 39, 209–215. Liu Y, Lv X, Tan R, Liu T, Chen T, Li M, Liu Y, Nie F, Wang X, Zhou P, 2014: A modified TALEN-based strategy for rapidly and efficiently generating knockout mice for kidney development studies. PLoS One 9, e84893. Luo X, Puig O, Hyun J, Bohmann D, Jasper H, 2006: Foxo and Fos regulate the
decision between cell death and survival in response to UV irradiation. EMBO J 26, 380–390. Ma S, Zhang S, Wang F, Liu Y, Liu Y, Xu H, Liu C, Lin Y, Zhao P, Xia Q, 2012: Highly efficient and specific genome editing in silkworm using custom TALENs. PLoS One 7, e45035. Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, Eichtinger M, Jiang T, Foley JE, Winfrey RJ, Townsend JA, 2008: Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell 31, 294–301. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM, 2013: RNA-guided human genome engineering via Cas9. Science 339, 823–826. Matsumoto M, Pocai A, Rossetti L, DePinho RA, Accili D, 2007: Impaired regulation of hepatic glucose production in mice lacking the forkhead transcription factor Foxo1 in liver. Cell Metab 6, 208–216. McManus MT, Sharp PA, 2002: Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 3, 737–747. Medema RH, Kops GJ, Bos JL, Burgering BM, 2000: AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature 404, 782–787. Modur V, Nagarajan R, Evers BM, Milbrandt J, 2002: FOXO proteins regulate tumor necrosis factor-related apoptosis inducing ligand expression Implications for PTEN mutation in prostate cancer. J Biol Chem 277, 47928–47937. Moghaddassi S, Eyestone W, Bishop CE, 2014: TALEN-mediated modification of the bovine genome for large-scale production of human serum albumin. PLoS One 9, e89631. Moniruzzaman M, Lee J, Zengyo M, Miyano T, 2010: Knockdown of FOXO3 induces primordial oocyte activation in pigs. Reproduction 139, 337–348. Moscou MJ, Bogdanove AJ, 2009: A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501. Nakagawa Y, Yamamoto T, Suzuki K, Araki K, Takeda N, Ohmuraya M, Sakuma T, 2014: Screening methods to identify TALEN-mediated knockout mice. Exp Anim 63, 79–84. Ogg S, Paradis S, Gottlieb S, Patterson GI, Lee L, Tissenbaum HA, Ruvkun G, 1997: The Fork head transcription factor DAF16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389, 994–999. Paik J-H, Kollipara R, Chu G, Ji H, Xiao Y, Ding Z, Miao L, Tothova Z, Horner JW, Carrasco DR, 2007: FoxOs are lineagerestricted redundant tumor suppressors and regulate endothelial cell homeostasis. Cell 128, 309–323. Panda SK, Wefers B, Ortiz O, Floss T, Schmid B, Haass C, Wurst W, K€ uhn R, 2013: Highly efficient targeted mutagenesis in mice using TALENs. Genetics 195, 703–713.
© 2015 Blackwell Verlag GmbH
Foxo3 Targeted Mice with TALENs Prosser HM, Koike-Yusa H, Cooper JD, Law FC, Bradley A, 2011: A resource of vectors and ES cells for targeted deletion of microRNAs in mice. Nat Biotechnol 29, 840–845. Ramirez CL, Foley JE, Wright DA, M€ ullerLerch F, Rahman SH, Cornu TI, Winfrey RJ, Sander JD, Fu F, Townsend JA, 2008: Unexpected failure rates for modular assembly of engineered zinc fingers. Nat Methods 5, 374–375. Sajwan S, Takasu Y, Tamura T, Uchino K, Sezutsu H, Zurovec M, 2013: Efficient disruption of endogenous Bombyx gene by TAL effector nucleases. Insect Biochem Mol Biol 43, 17–23. Sander JD, Dahlborg EJ, Goodwin MJ, Cade L, Zhang F, Cifuentes D, Curtin SJ, Blackburn JS, Thibodeau-Beganny S, Qi Y, 2011: Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). Nat Methods 8, 67–69. Sun N, Bao Z, Xiong X, Zhao H, 2014: SunnyTALEN: a second-generation TALEN system for human genome editing. Biotechnol Bioeng 111, 683–691.
© 2015 Blackwell Verlag GmbH
483 Tatsumi Y, Takeda M, Matsuda M, Suzuki T, Yokoi H, 2014: TALEN-mediated mutagenesis in zebrafish reveals a role for r-spondin 2 in fin ray and vertebral development. FEBS Lett 588, 4543–4550. Tran H, Brunet A, Grenier JM, Datta SR, Fornace AJ, DiStefano PS, Chiang LW, Greenberg ME, 2002: DNA repair pathway stimulated by the forkhead transcription factor FOXO3a through the Gadd45 protein. Science 296, 530–534. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD, 2010: Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11, 636–646. Watanabe T, Ochiai H, Sakuma T, Horch HW, Hamaguchi N, Nakamura T, Bando T, Ohuchi H, Yamamoto T, Noji S, 2012: Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases. Nat Commun 3, 1017. Wei C, Liu J, Yu Z, Zhang B, Gao G, Jiao R, 2013: TALEN or Cas9–rapid, efficient and specific choices for genome modifications. J Genet Genomics 40, 281–289.
Yang C, Tong H, Ma X, Du W, Liu D, Yang Y, Yan Y, 2014: Myostatin knockout in bovine fetal fibroblasts by using TALEN. Yi Chuan, 36, 685–690. Zhang Z, Xiang D, Heriyanto F, Gao Y, Qian Z, Wu W-S, 2013: Dissecting the roles of miR-302/367 cluster in cellular reprogramming using TALE-based repressor and TALEN. Stem Cell Rep 1, 218–225.
Submitted: 10 Nov 2014; Accepted: 23 Feb 2015 Author’s address (for correspondence): B Huang and D Shi, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, Guangxi 530005, China. E-mails: [email protected] (BH); [email protected] (DS)
Generation of Foxo3-targeted Mice by Injection of mRNAs Encoding Transcription Activator-like Effector Nucleases (TALENs) into Zygotes P Zhu1,*, Q Liu1,*, S Liu1, X Su1, W Feng1, X Lei1, J Liu1, K Cui1, B Huang1,2 and D Shi1 1 State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, Guangxi, China; 2Guangxi Experiment Centre of Science and Technology, Guangxi University, Nanning, Guangxi, China
Contents In this study, for exploring the mechanism of forkhead box O3 (Foxo3) participating in regulation of the activation of primordial oocytes, Foxo3-targeted mice were generated by injection of mRNAs encoding transcription activator-like effector nucleases (TALENs) into mouse zygotes. The TALEN sites were designed with high conservative homologous region among pig, bovine, buffalo and mouse by commercial biocompanies. The TALENs mutagenic non-homologous endjoining (NHEJ) repair activity were determined to be 31.3% in human embryonic kidney 293T (HEK-293T) cells by dual luciferase reporter assay system. Then, we firstly injected TALEN-mRNAs into the cytoplasm of mouse zygotes by micromanipulation, and four of 48 mouse blastocysts were identified as mutation by sequencing. Subsequently, by the method of TALEN-mRNAs injected into the zygotes with pronucleus micromanipulation technique, we obtained seven Foxo3 mutants of 20 FVB/NJ backgrounds mice which were Foxo3-independent alleles with frameshift and deletion mutations. It was very interesting that all seven were heterozygous mutants (Foxo3/+), and the gene mutagenesis rates of the mice reached 35%. The five Foxo3 mutant females were all infertile in the following 6 months after birth. The histological examination results showed that there were rare primordial follicles and primary follicles in the ovary of Foxo3 mutant compared to that of wide-type female mice. Moreover, one of two mutant males was subfertile and another was fertile normally. Those results suggested that the mutant of Foxo3 severely affected the fertile ability of female and perhaps male in some degree; furthermore, an even more efficient TALENsbased gene mutation method has been established to be poised to revolutionize the study of mouse and other species genetics.
Introduction The ability to precisely modify the mouse genome experimentally has had a considerable impact over the last 25 years in diverse areas of biomedical research and has made the mouse one of the most important model organisms in the laboratory today. Alterations in the genome are conventionally made by the process of gene targeting in embryonic stem cell via homologous recombination (Doyle et al. 2012). Using homologous recombination, whole genes or exons can be deleted from the mouse genome and the phenotypic consequences of these knockout models can deliver important information concerning gene function. However, the
*Those authors contributed to this article equivalently.
generation of knockout animals via the traditional method, such as homologous recombination, is a timeconsuming and laborious process, and therefore, a more-efficient tool is preferred for generating knockout mouse. Targeted gene knockdown by RNAi has provided researchers with a rapid, inexpensive and highthroughput alternative to homologous recombination (McManus and Sharp 2002). Yet, knockdown by RNAi is incomplete, varies between experiments and laboratories, has unpredictable off-target effects and provides temporary inhibition of gene function or can be permanently knockdown using transgenic approach in vivo or lentiviral vectors in vitro. In the past decade, a new approach has emerged that enables investigators to manipulate virtually any gene in a diverse range of cell types and organisms. The versatility of this approach is facilitated by the programmability of the DNA-binding domains that are derived from zinc-finger and transcription activator-like effector (TALE) proteins. This combination of simplicity and flexibility has catapulted zinc-finger nucleases (ZFNs) (Urnov et al. 2010; Carroll 2011) and transcription activator-like effector nucleases (TALENs)( Ikmi et al. 2014; Moghaddassi et al. 2014; Sun et al. 2014). Also, in the past years, a new approach has been developed and applied, it is just the clustered regulatory interspaced short palindromic repeats CRISPR/Cas (CRISPR-associated)-based RNA-guided DNA endonucleases (Cho et al. 2013; Cong et al. 2013; Mali et al. 2013). Up to now, these engineered TAL effector nucleases (TALENs) have been successfully applied to disrupt gene function in the yeast (Li et al. 2011), rice (Li et al. 2014), zebrasfish (Tatsumi et al. 2014), bovine (Yang et al. 2014), mouse (Nakagawa et al. 2014), human (Fadel et al. 2014) and so on. Knocking-out genes contain both protein coding genes (Bradley et al. 2012) and microRNA (Prosser et al. 2011; Zhang et al. 2013). Foxo3 is a member of the FOXO (forkhead box class O) transcription factors family and consists of four exons and produce a protein of a 673 amino acids. It has roles in cell cycle arrest (Medema et al. 2000; Alvarez et al. 2001), apoptosis (Dijkers et al. 2000; Modur et al. 2002) and DNA repair (Tran et al. 2002; Luo et al. 2006) among other functions. Consistent with their ability to block cell growth, inactivation of FOXO proteins is thought to be a critical event in oncogenic © 2015 Blackwell Verlag GmbH
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transformation (Greer and Brunet 2005; Fu and Tindall 2008). Deletion of all FOXO1, Foxo3 and FOXO4 alleles in adult mice induced a cancer prone phenotype, supporting a tumour-suppressing function of these proteins (Paik et al. 2007). In addition to their role in cell cycle regulation, FOXO transcription factors also regulate glucose metabolism in various organs (Matsumoto et al. 2007; Haeusler et al. 2010) and increase the resistance to oxidative stress (Kops et al. 2002; Brunet et al. 2004). Furthermore, the FOXO orthologue DAF-16 controls lifespan extension in Caenorhabditis elegans together with homologues of the human insulin receptor and the phosphatidylinositol 3-kinase (DAF-2 and AGE-1) (Lin et al. 1997; Ogg et al. 1997). Specifically Foxo3/ female mice had a very singular phenotype which they were initially fertile and subsequently infertile after 15 weeks of age (Castrillon et al. 2003; Hosaka et al. 2004). The activation of primordial follicle was presented in Foxo3 knockdown pigs (Moniruzzaman et al. 2010), but the mechanism remains to be further clarified. In this study, we were keen to produce Foxo3 knockdown mice by TALEN-based targeting technique and, consequently, able to establish an efficient genetargeting system for creating heritable mutations to apply for the study of mouse and other species genetics.
Materials and methods TALENs and reporters Plasmids encoding the TALENs pCS2-peas-T and reporter pSSA-LUC used in this study were obtained from viewsolid biotech (Beijing, China). Reporter pRGS were prepared as previously described (Kim et al. 2011). Briefly, oligonucleotides that contained target sites were synthesized (Sangon biotech, Shanghai, China) and annealed in vitro. The annealed oligonucleotides were ligated into the pRGS and pSSA-LUC (Table 1). After DNA for each of the Foxo3-TALEN and reporter plasmids were prepared using the Qiagen Endo-free Maxi-prep kit as directed by the manufacturer, reporter plasmid co-transfection of the Foxo3-TALEN vectors into HEK293T cells led to nuclease-dependent gene repair and enhanced green fluorescent protein (eGFP) and luciferase were expressed.
Cell culture and transfection HEK293T cells were maintained in Dulbecco’s modified Eagle medium (DMEM, Invitrogen) supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin, and 10% foetal bovine serum. Cells were transfected using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) at a weight ratio of 1 : 1 : 2 (plasmid encoding a TALEN: plasmid encoding the other TALEN: pRGS reporter) or 100 ng plasmid encoding a TALEN: 100 ng plasmid encoding the other TALEN: 50 ng pSSA-LUC reporter: 10 ng pRL-TK (Promega, Beijing, China). Dual luciferase reporter assay The 293T cells were seeded in 24-well plates. After 24-h incubation, cells were transfected with TALENs and pSSA-Luc (Sajwan et al. 2013; Aryan et al. 2013). Fortyeight hours after transfection, the cells were assayed by both firefly and renilla luciferase using the dual luciferase assay system (Promega) according to manufacturer’s instructions. All transfection experiments were conducted in triplicate and repeated three times independently. Preparation and cytoplasm or pronuclear microinjection of TALEN mRNA The mRNAs were generated with T7 RNA polymerase from 1 lg of linearized pCS2-peas-T construct using the mMessage mMachine T7 Kit (Life Technologies, 2013, Guangzhou, China) and purified using LiCl according to the manufacturer’s instructions. Purified mRNAs were diluted to 50 ng/ll for cytoplasm microinjection) or 100 ng/ll for pronuclear microinjection in 1 mM Tris.HCl pH 7.5/0.1 mM EDTA and were microinjected into the cytoplasm of fertilized oocytes. A pair of primers including Ftln-F and Ftln-R were designed and used to amplify the region containing the Foxo3 target site of the injected zygotes (Table 1). The PCR products were sequenced in BGI (Shenzhen, China), and gene mutation was analysed. Generation of Foxo3-targeted mice and genotyped Foxo3 knockout mice were generated by pronucleus microinjection TALEN-mRNAs in Cyagen (Guangzhou, China). Genomic DNA was isolated from tail tips of
Table 1. Primers used in this study Products
Amplicon length(bp)
Frgs
/
Fluc
/
Ftln
653
Primer name
Sequences(50 ?30 )
Frgs-F Frgs-R Fluc-F Fluc-R Ftln-F Ftln-R
aattcCCTGTACGTGGCCCCTGCAGAGGCCGGAGCTGCAGGCGAGCCCGGCCAAGCCCTg gatccAGGGCTTGGCCGGGCTCGCCTGCAGCTCCGGCCTCTGCAGGGGCCACGTACAGGg gatcCCTGTACGTGGCCCCTGCAGAGGCCGGAGCTGCAGGCGAGCCCGGCCAAGCCCTg tcgaAGGGCTTGGCCGGGCTCGCCTGCAGCTCCGGCCTCTGCAGGGGCCACGTACAGGg CGGACTAGGCTCCAACAC ACCTGTCCTATGCCGACC
The annealed oligonucleotides Frgs and Fluc were ligated into the pRGS and pSSA-LUC, respectively. Primers Ftln was used to amplify the region containing the Foxo3 target site.
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founder mice. The primers Ftln-F and Ftln-R were used to amplify the region containing the Foxo3 target site from wild-type individuals or founders. The PCR products that included TALEN-target sites were purified using the Gel Extraction Kit (Tiangen, Beijing, China) and cloned into the pEASY-T-Blunt vector using the pEASYT-Blunt PCR Cloning Kit (Transgen, Beijing, China). Cloned plasmids were sequenced in BGI (Shenzhen, China), and gene mutation was analysed.
Histology Ovaries were fixed for 3–5 h in 2.5% glutaraldehyde and 2.5% paraformaldehyde in 0.083 M sodium cacodylate buffer, pH 7.2, 4°C. They were then washed in 0.1 M sodium cacodylate buffer, pH 7.2, for 24 h before embedding in JB-4 (glycol methacrylate) plastic (Polysciences, Inc., Warrington, PA, USA). Sections, 2 lm thick, were stained with haematoxylin and eosin.
(a)
(b)
(c)
Fig. 1. FOXO3 genomic structure, TALEN binding sites and report plasmids structure. (a) Genomic structure of the mouse FOXO3 (upper panel) with an enlargement of exon 2 (lower panel), showing the binding sites of for TALEN together with the binding sites of the PCR primers, Ftln-F and Ftln-R used to genotype the mutant alleles. The FOXO3 TALEN target sequence, with Left and Right TALEN monomer binding sites underlined were shown. The TALEN sites were high conservative homologous region among pig, bovine, buffalo and mouse. (b) Outline of the nuclease reporter pRGS assay. TALEN-FOXO3 targeted sequences were cloned into the locations between the report gene RFP and eGFP, Reporter plasmid co-transfection of the TALEN-FOXO3 vectors into HEK293T cells led to nuclease dependent gene repair and eGFP was expressed, as shown by green fluorescence. The red fluorescence from RFP was used as an indicator of plasmid transfection efficiency. (c) Outline of the nuclease reporter pSSA-LUC assay. TALEN-FOXO3 targeted sequences were cloned into the locations between the luciferase gene segments. Reporter plasmid co-transfection of the TALEN-FOXO3 vectors and pRL-TK into HEK293T cells led to nuclease dependent gene repair and luciferase was expressed. The renilla fluorescence was used as an indicator of plasmid transfection efficiency
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482 factors. Proc Natl Acad Sci 97, 1495– 1500. Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U, 2009: Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509–1512. Bradley A, Anastassiadis K, Ayadi A, Battey JF, Bell C, Birling M-C, Bottomley J, Brown SD, B€ urger A, Bult CJ, 2012: The mammalian gene function resource: the international knockout mouse consortium. Mamm Genome 23, 580–586. Brinster RL, Chen HY, Trumbauer ME, Yagle MK, Palmiter RD, 1985: Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc Natl Acad Sci 82, 4438–4442. Brunet A, Sweeney LB, Sturgill JF, Chua KF, Greer PL, Lin Y, Tran H, Ross SE, Mostoslavsky R, Cohen HY, 2004: Stressdependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science 303, 2011–2015. Cade L, Reyon D, Hwang WY, Tsai SQ, Patel S, Khayter C, Joung JK, Sander JD, Peterson RT, Yeh J-RJ, 2012: Highly efficient generation of heritable zebrafish gene mutations using homo-and heterodimeric TALENs. Nucleic Acids Res 40, 8001–8010. Carlson DF, Tan W, Lillico SG, Stverakova D, Proudfoot C, Christian M, Voytas DF, Long CR, Whitelaw CBA, Fahrenkrug SC, 2012: Efficient TALEN-mediated gene knockout in livestock. Proc Natl Acad Sci 109, 17382–17387. Carroll D, 2011: Genome engineering with zinc-finger nucleases. Genetics 188, 773–782. Castrillon DH, Miao L, Kollipara R, Horner JW, DePinho RA, 2003: Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a. Science 301, 215–218. Chen S, Oikonomou G, Chiu CN, Niles BJ, Liu J, Lee DA, Antoshechkin I, Prober DA, 2013: A large-scale in vivo analysis reveals that TALENs are significantly more mutagenic than ZFNs generated using context-dependent assembly. Nucleic Acids Res 41, 2769–2778. Cho SW, Kim S, Kim JM, Kim J-S, 2013: Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31, 230–232. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, 2013: Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823. Dijkers PF, Lammers J-WJ, Koenderman L, Coffer PJ, 2000: Expression of the proapoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1. Curr Biol 10, 1201– 1204. Doyle A, McGarry MP, Lee NA, Lee JJ, 2012: The construction of transgenic and gene knockout/knockinmousemodels ofhuman disease. Transgenic Res 21, 327–349. Fadel HJ, Morrison JH, Saenz DT, Fuchs JR, Kvaratskhelia M, Ekker SC, Poeschla
P Zhu, Q Liu, S Liu, X Su, W Feng, X Lei, J Liu, K Cui, B Huang and D Shi EM, 2014: TALEN knockout of the PSIP1 gene in human cells: analyses of HIV-1 replication and allosteric integrase inhibitor mechanism. J Virol 88, 9704– 9717. Fu Z, Tindall D, 2008: FOXOs, cancer and regulation of apoptosis. Oncogene 27, 2312–2319. Geurts AM, Cost GJ, Freyvert Y, Zeitler B, Miller JC, Choi VM, Jenkins SS, Wood A, Cui X, Meng X, 2009: Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325, 433–433. Greer EL, Brunet A, 2005: FOXO transcription factors at the interface between longevity and tumor suppression. Oncogene 24, 7410–7425. Haeusler RA, Kaestner KH, Accili D, 2010: FoxOs function synergistically to promote glucose production. J Biol Chem 285, 35245–35248. Hosaka T, Biggs WH, Tieu D, Boyer AD, Varki NM, Cavenee WK, Arden KC, 2004: Disruption of forkhead transcription factor (FOXO) family members in mice reveals their functional diversification. Proc Natl Acad Sci USA 101, 2975– 2980. Ikmi A, McKinney SA, Delventhal KM, Gibson MC, 2014: TALEN and CRISPR/ Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis. Nat Commun 5, 5486. Kim H, Um E, Cho S-R, Jung C, Kim H, Kim J-S, 2011: Surrogate reporters for enrichment of cells with nucleaseinduced mutations. Nat Methods 8, 941– 943. Kops GJ, Dansen TB, Polderman PE, Saarloos I, Wirtz KW, Coffer PJ, Huang T-T, Bos JL, Medema RH, Burgering BM, 2002: Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 419, 316–321. Li T, Huang S, Jiang WZ, Wright D, Spalding MH, Weeks DP, Yang B, 2011: TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res 39, 359–372. Li T, Liu B, Chen CY, Yang B, 2014: TALEN utilization in rice genome modifications. Methods 69, 9–16. Lin K, Dorman JB, Rodan A, Kenyon C, 1997: daf-16: an HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278, 1319–1322. Liu J, Li C, Yu Z, Huang P, Wu H, Wei C, Zhu N, Shen Y, Chen Y, Zhang B, 2012: Efficient and specific modifications of the drosophila genome by means of an easy TALEN strategy. J Genet Genomics 39, 209–215. Liu Y, Lv X, Tan R, Liu T, Chen T, Li M, Liu Y, Nie F, Wang X, Zhou P, 2014: A modified TALEN-based strategy for rapidly and efficiently generating knockout mice for kidney development studies. PLoS One 9, e84893. Luo X, Puig O, Hyun J, Bohmann D, Jasper H, 2006: Foxo and Fos regulate the
decision between cell death and survival in response to UV irradiation. EMBO J 26, 380–390. Ma S, Zhang S, Wang F, Liu Y, Liu Y, Xu H, Liu C, Lin Y, Zhao P, Xia Q, 2012: Highly efficient and specific genome editing in silkworm using custom TALENs. PLoS One 7, e45035. Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, Eichtinger M, Jiang T, Foley JE, Winfrey RJ, Townsend JA, 2008: Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell 31, 294–301. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM, 2013: RNA-guided human genome engineering via Cas9. Science 339, 823–826. Matsumoto M, Pocai A, Rossetti L, DePinho RA, Accili D, 2007: Impaired regulation of hepatic glucose production in mice lacking the forkhead transcription factor Foxo1 in liver. Cell Metab 6, 208–216. McManus MT, Sharp PA, 2002: Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 3, 737–747. Medema RH, Kops GJ, Bos JL, Burgering BM, 2000: AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature 404, 782–787. Modur V, Nagarajan R, Evers BM, Milbrandt J, 2002: FOXO proteins regulate tumor necrosis factor-related apoptosis inducing ligand expression Implications for PTEN mutation in prostate cancer. J Biol Chem 277, 47928–47937. Moghaddassi S, Eyestone W, Bishop CE, 2014: TALEN-mediated modification of the bovine genome for large-scale production of human serum albumin. PLoS One 9, e89631. Moniruzzaman M, Lee J, Zengyo M, Miyano T, 2010: Knockdown of FOXO3 induces primordial oocyte activation in pigs. Reproduction 139, 337–348. Moscou MJ, Bogdanove AJ, 2009: A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501. Nakagawa Y, Yamamoto T, Suzuki K, Araki K, Takeda N, Ohmuraya M, Sakuma T, 2014: Screening methods to identify TALEN-mediated knockout mice. Exp Anim 63, 79–84. Ogg S, Paradis S, Gottlieb S, Patterson GI, Lee L, Tissenbaum HA, Ruvkun G, 1997: The Fork head transcription factor DAF16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389, 994–999. Paik J-H, Kollipara R, Chu G, Ji H, Xiao Y, Ding Z, Miao L, Tothova Z, Horner JW, Carrasco DR, 2007: FoxOs are lineagerestricted redundant tumor suppressors and regulate endothelial cell homeostasis. Cell 128, 309–323. Panda SK, Wefers B, Ortiz O, Floss T, Schmid B, Haass C, Wurst W, K€ uhn R, 2013: Highly efficient targeted mutagenesis in mice using TALENs. Genetics 195, 703–713.
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to measure activity in mammalian cells HEK293T, repair activity was analysed using SPSS version 19.0 (SPSS, Inc., Chicago, IL, USA), and a p-value of less than 0.05 was considered to be significant.
Results Selection of TALEN-target site and activity assay in vitro For exploring the possibility of target same site of Foxo3 gene with different species, TALEN sites were designed for Foxo3 which were homologous among pig, bovine, buffalo and mouse (Fig. 1a). TALEN-Foxo3 construct was cloned into a mammalian expression vector and functionally validated using pRGS reporter assay or pSSA-LUC reporter assay in HEK293T cells. Firstly, we used pRGS reporter to investigate whether this TALEN can cleave the target sequence incorporated between the RFP and GFP sequences in HEK293T cells (Fig. 1b). In this reporter, the gfp sequence was fused to the end of rfp sequence and inactivated by frameshift mutation and only expressed when the target sequence was cleaved by site-specific nucleases, which caused some small, frameshifting
insertions or deletions around the target sequence by means of error-prone non-homologous end-joining repair of the double-strand breaks. In this study, Foxo3-TALENs can recognize and cleave the target DNA sequence (Fig. 2a–c) demonstrated by GFP-expressing cells only obtained from the treatment of co-transfection with the in vitrotranscribed Foxo3-TALENs. Then, we used a pSSLUC reporter to measure their activity in mammalian cells. Site-specific nucleases were fused to the luciferase sequence (Fig. 1c). Luciferase is expressed only when the target sequence is cleaved by site-specific nucleases. By co-transfecting TALENS, pSS-LUC and renilla, we found that the TALENS mutagenic nonhomologous end-joining repair efficiency reached 31.3% (Fig. 2d). Generation and identification of Foxo3 mutant mice Firstly, in vitro-transcribed Foxo3-TALENs mRNAs were injected into the cytoplasm of fifty-five mouse zygotes by micromanipulation, and subsequently, those injected zygotes were in vitro cultured into blastocyst
(a)
(b)
Fig. 3. PCR and genotypic analysis revealed mutations in 4 mouse blastocyst injected with FOXO3 TALENs. (a) Blastocyst 52 and 30 were heterozygous, 46 and 53 were deleted 52 bp and 88 bp. (b) No mutations in others blastocyst
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Foxo3 Targeted Mice with TALENs
479
(a)
(b)
(c)
Fig. 4. Created FOXO3 knockout mice by TALEN-mediated gene targeting and sequence information of FOXO3 mutant alleles. (a) Seven maturation Founders by TALEN-mediated gene targeting. (b) Mutations of 20 pups by PCR detecting. (c) Sequences of FOXO3 mutations in seven F0 mice lines. Deletions are indicated by dashes with gray highlight and insertions are highlighted in gray. The DNA sequences to which the TALEN monomers were designed are highlighted in red. (d) DNA sequencing result of seven F0 mice lines. (e) To determine whether the FOXO3 mutations were heritable, we screened all the founders and found that the two male mice transmitted target site mutations to their F1 mice, demonstrating successful germline transmission. 1–10, F1 female mice of NO.5 F0 male, 11–19, F1 male mice of NO.5 F0 male, 20, F1 mouse of NO.4 F0 male, 21, NO.4 F0 male, 22, NO.5 F0 male
(d)
(e)
stage. The following PCR and genotypic analysis results indicated that there were four mutants among 48 analysed embryos (Fig. 3a,b). © 2015 Blackwell Verlag GmbH
Secondly, pronuclear microinjections of TALENFoxo3 mRNAs (50 ng/ll) were conducted. Then, the pups were screened for the presence of mutant alleles by
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sequence analysis of PCR products covering the targeted exon. Seven of 20 mice (35%) were identified as Foxo3 heterozygous mutant (Foxo3/+) (Fig. 4, Table 2).Thirdly, sequence alignment results showed that the Foxo3 sequences of two male mice were deleted 34 or 51 bp, respectively. Those of the other female were added 2 bp, or deleted 15, 47 or 100 bp, respectively. Phenotypes observation of Foxo3 mutant mice To determine whether the Foxo3 mutations were heritable, two mutant female mice were caged with one mutant male for 2 weeks. And the other mutant female was caged with a wide-type male for 2 weeks. Unfortunately, none mutant female was pregnant. Therefore, we arranged the two mutant males (Nos. 4 and 5) to cage with two wide-type female, respectively; subsequently, the two female all got pregnancy. No.5 mutant male produced normal number of pups compared to that of wild type; however, No.4 mutant male only got six pups (Table 3) during a long caged period. The ovary of mutant (Foxo3+/) female mice was collected, and no corpus luteum was observed, which maybe suggest Foxo3 knockdown would disrupt ovulation. The histological examination results showed there were rare primordial and early follicles were observed compared to that of wide-type female (Fig. 5).
Discussion With the development of new and affordable methods for whole-genome sequencing, scientists are poised to deliver upon the promises of the genomic revolution to transform basic science and personalized medicine. However, the use of homologous recombination has been hampered by several factors, including the low efficiency at which engineered constructs are correctly inserted into the chromosomal target site, the need for time-consuming and labour-insensitive selection/screening strategies, and the potential for adverse mutagenic effects. While current RNAi technology has enabled to increase the efficiency of homologous recombination, it is still limited by variation in the degree and timing of knockdown, and off-target effects. In contrast, the
Table 3. The reproductive ability of NO.4 male mouse Female and age 1 2 3 4 5 6
(1.5 (1.5 (1.5 (1.5 (1.5 (1.5
month) month) month) month) month) month)
Age of NO.4 male
Caged time
Born
1.5 month 1.5 month 1.5 month 3 month 3 month 3 month
Caged for 1.5 month
3 3 0 0 0 0
Caged for 2 month
No.4 mutant male got six pups which suggested it maybe subfertile.
ability to directly and specifically disrupt a gene of interest offers the possibility to perform intricate reverse genetic experiments on any gene, in any organism. In the past decade, zinc-finger nucleases (Beerli et al. 1998, 2000) and transcription activator-like effector nucleases (Boch et al. 2009; Moscou and Bogdanove 2009) had been developed and applicated. They had been successfully applied to disrupt gene function in different organism with high editing rates. A major drawback of ZFNs, however, is the elaborate and time-consuming experimental selection process (Maeder et al. 2008). Although simplified methods, such as modular assembly and CoDA, have been reported (Sander et al. 2011), the quality of ZFNs generated by such platforms is controversial (Ramirez et al. 2008) or not determined yet. In zebrafish, TALENs are proved significantly more likely to be mutagenic and induce an average of 10-fold more mutations than ZFNs, 20% and 2% for TALENs and ZFNs, respectively (Chen et al. 2013). Up to date, in the family of genomic editing toolbox, TALEN has shown to be an efficient, rapid, specific and economic method with a wide range of applications. TALEN-induced indels are often variable in length, often leading to a frameshift. It is worth noting that different pairs of TALENs yield indels with different efficiencies, ranging from 0% to approximately 65% in flies and 0% to approximately 50% in zebrafish (Wei et al. 2013). In other laboratories, seven TALENs targets in various endogenous zebrafish genes induced targeted indel mutations with high efficiencies ranging from 2% to 76% (Cade et al. 2012). DF Carlson found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in
Table 2. The presence of mutant alleles of 7 mice Mouse
Gender
4 5 6 8 12 17 18
Male Male Female Female Female Female Female
Mutations Delete 47 bp Delete 51 bp Added 2 bp Delete 15 bp Delete 34 bp Delete 100 bp Not detected by plasmids sequencing picks by PCR products sequencing
Change protein Out of frame Delete 17 aa Out of frame Delete 5 aa Out of frame Out of frame but was mixed
Phenotypes Subfertile Normal reproduction Infertile Infertile Infertile Infertile Infertile
The presence of mutant alleles by sequence analysis of PCR products covering the targeted exon, and seven of these mice (35%) were identified as founders harboring mutant Foxo3 alleles.
© 2015 Blackwell Verlag GmbH
Foxo3 Targeted Mice with TALENs
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(a)
(b)
Fig. 5. Histological analysis of Foxo3a–/+ and control ovaries at 20 weeks. (a) Histology of widetype mouse ovary. (b) Histology of number 12 female founder ovary. Green arrows: small follicles
primary cells. Cytoplasmic injections of TALENmRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analysed (Carlson et al. 2012). In our experiment, the success rate we observed is 35% (>20%), similar to those described in many other organisms (Liu et al. 2012; Ma et al. 2012; Panda et al. 2013). And the TALEN-based editing was associated with deletions in size 47, 51 and 100 bp, respectively, which are longer than others report of small detections ranging in size from 1 to 7 bp (Liu et al. 2012; Ma et al. 2012; Watanabe et al. 2012). In addition, large deletions and inversions of sequences that are longer than 6 kb have been obtained in pigs by targeting two TALEN pairs to the same chromosome (Carlson et al. 2012). In our study, mutations in blastocysts generated by cytoplasm microinjections (8.3%) were lower than that of generated by pronucleus microinjection (35%). However, Liu et al. (2014) demonstrate that cytoplasm microinjection may result in a higher efficiency (85.7%) than that of pronucleus microinjection (30.4%). In 1985, Brinster (Brinster et al. 1985) observed that the integration frequency of linearized DNA injected in the cytoplasm or male pronucleus of mouse zygotes was 3.4% or 25–30%, respectively. Geurts (Geurts et al. 2009) revealed that rate of successful mutagenesis of lgM gene by pronucleus or cytoplasm microinjection ZFN mRNAs into the zygotes was 13–16% or 2–13% in rat, respectively. The reasons for different abilities of DNA or mRNA function after cytoplasm or pronucleus injection maybe caused by strain, gene or proficiency in injection. Foxo3/ female mice have a very singular phenotype which are initially fertile and subsequently infertile after 15 weeks of age (Castrillon et al. 2003; Hosaka et al. 2004). Foxo3 knockdown induced primordial oocyte activation in pigs (Moniruzzaman et al. 2010). In our study, all Foxo3 gene knockout females were infertile,
References Alvarez B, Martınez-A C, Burgering BM, Carrera AC, 2001: Forkhead transcription factors contribute to execution of the mitotic programme in mammals. Nature 413, 744–747.
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and their primordial oocytes were rare. The two mutant males, one of them in which the Foxo3 sequence was deleted 51 bp was normal fertile; however, another was subfertile in which Foxo3 sequence was deleted 34 bp. It maybe indicate that Foxo3 influence male reproduction, but more researches should be conducted to determine this point.. In conclusion, our study establishes that the TALENmediated targeting is an efficient method for creating heritable mutations in Foxo3 exon2 locus of the mouse genome, and also, this TALEN sites are homologous to bovine and sus, the work using these TALENs to edit bovine or sus genome is being in progress (data not shown). Taken together, these data suggest that TALEN-mediated in vivo mutagenesis might expedite the creation of genetically engineered mouse models or livestock and thereby help to accelerate functional genomic research. Acknowledgements This work was funded by the China Transgenic Project (2011ZX08007-003) and the National Natural Science Fund (Grant No. 31160457 and 31401267), the Scientific Research Foundation of Guangxi University (Grant No. XGZ130880), Guangxi Natural Science Foundation (Grant No. 2014GXNSFCB118003), and Guangxi Experiment Centre of Science and Technology (YXKT2014005).
Conflict of interest None of the authors have any conflict of interest to declare.
Author contributions Peng Zhu and Qingyou Liu designed and conducted the experiment, Shuai Liu, Xiaoping Su, Wanyou Feng, Xiaocan Lei, Kuiqing Cui and Jinfeng Liu conducted part of experiments, Ben Huang and Deshun Shi analysed data and drafted manuscript.
Aryan A, Anderson MA, Myles KM, Adelman ZN, 2013: TALEN-based gene disruption in the dengue vector Aedes aegypti. PLoS One 8, e60082. Beerli RR, Segal DJ, Dreier B, Barbas CF, 1998: Toward controlling gene expression at will: specific regulation of the erbB-2/
HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. Proc Natl Acad Sci 95, 14628–14633. Beerli RR, Dreier B, Barbas CF, 2000: Positive and negative regulation of endogenous genes by designed transcription
482 factors. Proc Natl Acad Sci 97, 1495– 1500. Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U, 2009: Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509–1512. Bradley A, Anastassiadis K, Ayadi A, Battey JF, Bell C, Birling M-C, Bottomley J, Brown SD, B€ urger A, Bult CJ, 2012: The mammalian gene function resource: the international knockout mouse consortium. Mamm Genome 23, 580–586. Brinster RL, Chen HY, Trumbauer ME, Yagle MK, Palmiter RD, 1985: Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc Natl Acad Sci 82, 4438–4442. Brunet A, Sweeney LB, Sturgill JF, Chua KF, Greer PL, Lin Y, Tran H, Ross SE, Mostoslavsky R, Cohen HY, 2004: Stressdependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science 303, 2011–2015. Cade L, Reyon D, Hwang WY, Tsai SQ, Patel S, Khayter C, Joung JK, Sander JD, Peterson RT, Yeh J-RJ, 2012: Highly efficient generation of heritable zebrafish gene mutations using homo-and heterodimeric TALENs. Nucleic Acids Res 40, 8001–8010. Carlson DF, Tan W, Lillico SG, Stverakova D, Proudfoot C, Christian M, Voytas DF, Long CR, Whitelaw CBA, Fahrenkrug SC, 2012: Efficient TALEN-mediated gene knockout in livestock. Proc Natl Acad Sci 109, 17382–17387. Carroll D, 2011: Genome engineering with zinc-finger nucleases. Genetics 188, 773–782. Castrillon DH, Miao L, Kollipara R, Horner JW, DePinho RA, 2003: Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a. Science 301, 215–218. Chen S, Oikonomou G, Chiu CN, Niles BJ, Liu J, Lee DA, Antoshechkin I, Prober DA, 2013: A large-scale in vivo analysis reveals that TALENs are significantly more mutagenic than ZFNs generated using context-dependent assembly. Nucleic Acids Res 41, 2769–2778. Cho SW, Kim S, Kim JM, Kim J-S, 2013: Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31, 230–232. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, 2013: Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823. Dijkers PF, Lammers J-WJ, Koenderman L, Coffer PJ, 2000: Expression of the proapoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1. Curr Biol 10, 1201– 1204. Doyle A, McGarry MP, Lee NA, Lee JJ, 2012: The construction of transgenic and gene knockout/knockinmousemodels ofhuman disease. Transgenic Res 21, 327–349. Fadel HJ, Morrison JH, Saenz DT, Fuchs JR, Kvaratskhelia M, Ekker SC, Poeschla
P Zhu, Q Liu, S Liu, X Su, W Feng, X Lei, J Liu, K Cui, B Huang and D Shi EM, 2014: TALEN knockout of the PSIP1 gene in human cells: analyses of HIV-1 replication and allosteric integrase inhibitor mechanism. J Virol 88, 9704– 9717. Fu Z, Tindall D, 2008: FOXOs, cancer and regulation of apoptosis. Oncogene 27, 2312–2319. Geurts AM, Cost GJ, Freyvert Y, Zeitler B, Miller JC, Choi VM, Jenkins SS, Wood A, Cui X, Meng X, 2009: Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325, 433–433. Greer EL, Brunet A, 2005: FOXO transcription factors at the interface between longevity and tumor suppression. Oncogene 24, 7410–7425. Haeusler RA, Kaestner KH, Accili D, 2010: FoxOs function synergistically to promote glucose production. J Biol Chem 285, 35245–35248. Hosaka T, Biggs WH, Tieu D, Boyer AD, Varki NM, Cavenee WK, Arden KC, 2004: Disruption of forkhead transcription factor (FOXO) family members in mice reveals their functional diversification. Proc Natl Acad Sci USA 101, 2975– 2980. Ikmi A, McKinney SA, Delventhal KM, Gibson MC, 2014: TALEN and CRISPR/ Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis. Nat Commun 5, 5486. Kim H, Um E, Cho S-R, Jung C, Kim H, Kim J-S, 2011: Surrogate reporters for enrichment of cells with nucleaseinduced mutations. Nat Methods 8, 941– 943. Kops GJ, Dansen TB, Polderman PE, Saarloos I, Wirtz KW, Coffer PJ, Huang T-T, Bos JL, Medema RH, Burgering BM, 2002: Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 419, 316–321. Li T, Huang S, Jiang WZ, Wright D, Spalding MH, Weeks DP, Yang B, 2011: TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res 39, 359–372. Li T, Liu B, Chen CY, Yang B, 2014: TALEN utilization in rice genome modifications. Methods 69, 9–16. Lin K, Dorman JB, Rodan A, Kenyon C, 1997: daf-16: an HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278, 1319–1322. Liu J, Li C, Yu Z, Huang P, Wu H, Wei C, Zhu N, Shen Y, Chen Y, Zhang B, 2012: Efficient and specific modifications of the drosophila genome by means of an easy TALEN strategy. J Genet Genomics 39, 209–215. Liu Y, Lv X, Tan R, Liu T, Chen T, Li M, Liu Y, Nie F, Wang X, Zhou P, 2014: A modified TALEN-based strategy for rapidly and efficiently generating knockout mice for kidney development studies. PLoS One 9, e84893. Luo X, Puig O, Hyun J, Bohmann D, Jasper H, 2006: Foxo and Fos regulate the
decision between cell death and survival in response to UV irradiation. EMBO J 26, 380–390. Ma S, Zhang S, Wang F, Liu Y, Liu Y, Xu H, Liu C, Lin Y, Zhao P, Xia Q, 2012: Highly efficient and specific genome editing in silkworm using custom TALENs. PLoS One 7, e45035. Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, Eichtinger M, Jiang T, Foley JE, Winfrey RJ, Townsend JA, 2008: Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell 31, 294–301. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM, 2013: RNA-guided human genome engineering via Cas9. Science 339, 823–826. Matsumoto M, Pocai A, Rossetti L, DePinho RA, Accili D, 2007: Impaired regulation of hepatic glucose production in mice lacking the forkhead transcription factor Foxo1 in liver. Cell Metab 6, 208–216. McManus MT, Sharp PA, 2002: Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 3, 737–747. Medema RH, Kops GJ, Bos JL, Burgering BM, 2000: AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature 404, 782–787. Modur V, Nagarajan R, Evers BM, Milbrandt J, 2002: FOXO proteins regulate tumor necrosis factor-related apoptosis inducing ligand expression Implications for PTEN mutation in prostate cancer. J Biol Chem 277, 47928–47937. Moghaddassi S, Eyestone W, Bishop CE, 2014: TALEN-mediated modification of the bovine genome for large-scale production of human serum albumin. PLoS One 9, e89631. Moniruzzaman M, Lee J, Zengyo M, Miyano T, 2010: Knockdown of FOXO3 induces primordial oocyte activation in pigs. Reproduction 139, 337–348. Moscou MJ, Bogdanove AJ, 2009: A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501. Nakagawa Y, Yamamoto T, Suzuki K, Araki K, Takeda N, Ohmuraya M, Sakuma T, 2014: Screening methods to identify TALEN-mediated knockout mice. Exp Anim 63, 79–84. Ogg S, Paradis S, Gottlieb S, Patterson GI, Lee L, Tissenbaum HA, Ruvkun G, 1997: The Fork head transcription factor DAF16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389, 994–999. Paik J-H, Kollipara R, Chu G, Ji H, Xiao Y, Ding Z, Miao L, Tothova Z, Horner JW, Carrasco DR, 2007: FoxOs are lineagerestricted redundant tumor suppressors and regulate endothelial cell homeostasis. Cell 128, 309–323. Panda SK, Wefers B, Ortiz O, Floss T, Schmid B, Haass C, Wurst W, K€ uhn R, 2013: Highly efficient targeted mutagenesis in mice using TALENs. Genetics 195, 703–713.
© 2015 Blackwell Verlag GmbH
Foxo3 Targeted Mice with TALENs Prosser HM, Koike-Yusa H, Cooper JD, Law FC, Bradley A, 2011: A resource of vectors and ES cells for targeted deletion of microRNAs in mice. Nat Biotechnol 29, 840–845. Ramirez CL, Foley JE, Wright DA, M€ ullerLerch F, Rahman SH, Cornu TI, Winfrey RJ, Sander JD, Fu F, Townsend JA, 2008: Unexpected failure rates for modular assembly of engineered zinc fingers. Nat Methods 5, 374–375. Sajwan S, Takasu Y, Tamura T, Uchino K, Sezutsu H, Zurovec M, 2013: Efficient disruption of endogenous Bombyx gene by TAL effector nucleases. Insect Biochem Mol Biol 43, 17–23. Sander JD, Dahlborg EJ, Goodwin MJ, Cade L, Zhang F, Cifuentes D, Curtin SJ, Blackburn JS, Thibodeau-Beganny S, Qi Y, 2011: Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). Nat Methods 8, 67–69. Sun N, Bao Z, Xiong X, Zhao H, 2014: SunnyTALEN: a second-generation TALEN system for human genome editing. Biotechnol Bioeng 111, 683–691.
© 2015 Blackwell Verlag GmbH
483 Tatsumi Y, Takeda M, Matsuda M, Suzuki T, Yokoi H, 2014: TALEN-mediated mutagenesis in zebrafish reveals a role for r-spondin 2 in fin ray and vertebral development. FEBS Lett 588, 4543–4550. Tran H, Brunet A, Grenier JM, Datta SR, Fornace AJ, DiStefano PS, Chiang LW, Greenberg ME, 2002: DNA repair pathway stimulated by the forkhead transcription factor FOXO3a through the Gadd45 protein. Science 296, 530–534. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD, 2010: Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11, 636–646. Watanabe T, Ochiai H, Sakuma T, Horch HW, Hamaguchi N, Nakamura T, Bando T, Ohuchi H, Yamamoto T, Noji S, 2012: Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases. Nat Commun 3, 1017. Wei C, Liu J, Yu Z, Zhang B, Gao G, Jiao R, 2013: TALEN or Cas9–rapid, efficient and specific choices for genome modifications. J Genet Genomics 40, 281–289.
Yang C, Tong H, Ma X, Du W, Liu D, Yang Y, Yan Y, 2014: Myostatin knockout in bovine fetal fibroblasts by using TALEN. Yi Chuan, 36, 685–690. Zhang Z, Xiang D, Heriyanto F, Gao Y, Qian Z, Wu W-S, 2013: Dissecting the roles of miR-302/367 cluster in cellular reprogramming using TALE-based repressor and TALEN. Stem Cell Rep 1, 218–225.
Submitted: 10 Nov 2014; Accepted: 23 Feb 2015 Author’s address (for correspondence): B Huang and D Shi, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, Guangxi 530005, China. E-mails: [email protected] (BH); [email protected] (DS)
