Journal of Fish Biology (2014) 85, 1369–1380 doi:10.1111/jfb.12487, available online at wileyonlinelibrary.com
Cell-penetrating peptide delivery of biologically active oct4 protein into cultured Takifugu rubripes spermary cells X. X. Yang*, X. N. Hou, B. Xu, X. Hao, G. J. Jiang and T. J. Fan Department of Marine Biology, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China
(Received 28 October 2013, Accepted 27 June 2014) Continuous cell culture of a puffer fish Takifugu rubripes has been established for efficient delivery of exogenous genes or proteins to cultured fish cells. Transcription factor oct4 was chosen for transduction into cultured fish cells because of its conserved structure and function between fish and mammals. In this work, the T. rubripes oct4 gene was cloned and expressed in Escherichia coli as a recombinant protein by introducing cell-penetrating peptide (CPP) poly-arginine (11R) and 6His-tag at the C-terminus. After purification, recombinant proteins were added to the growth medium and incubated with T. rubripes spermary cells. Recombinant proteins that crossed the cell membrane were detected in the cytoplasm and nucleus by western blot and immunofluorescent observation. The function of transduced oct4 as a transcription factor in fish cells was confirmed by driving green fluorescent protein expression in the pEGFP-1 reporter construct with the conserved specific oct4-binding sequence from mouse Mus musculus. Taken together, 11R can be an efficient CPP in delivering fusion proteins to cultured fish cells. © 2014 The Fisheries Society of the British Isles
Key words: 11R; cell culture; puffer fish; recombinant protein; transcription factor.
INTRODUCTION Fish cell cultures are of great importance for theoretical and practical fish biological and pathological research. Improvements of in vitro DNA transfection in fish cell lines can improve in vitro assays for heterologous fish cell gene expression (Hackett & Alvarez, 2000), advance fish DNA vaccination methods (Lorenzen et al., 2002; Sommerset et al., 2005; Kurath, 2008), allow the search for new promoters that are active in fish cells (Collet et al., 2004; Ruiz et al., 2008) and enhance studies of small interfering (si)RNA interference in permanently transformed fish cell lines (Ruiz et al., 2009). Fish cell transfection was first reported in the context of the calcium chloride method (Bearzotti et al., 1992), and this technique was later improved with commercially available liposomes such as Lipofectamine and Fugene 6 (Lopez et al., 2001; Ruiz et al., 2009). Generally, gene delivery efficiencies were low in fish cells compared to mammal cell cultures (Huang et al., 2011). *Author to whom correspondence should be addressed. Tel.: +86 532 82031793; email: [email protected]
1369 © 2014 The Fisheries Society of the British Isles
1370
X . X . YA N G E T A L.
For those genes that could be transduced successfully into fish cells, expression was restricted due to low promoter efficiency and varied transcriptional activity that depended on the cell type studied. Although human cytomegalovirus immediate early (CMV-IE) promoters can offer greater expression than promoters of most animal genes, these promoters are not equally functional in all cell types, especially in fish cells. Previously, Takifugu rubripes (Temminck & Schlegel 1850) oct4 was constructed into pcDNA3.1 under the control of the CMV-IE promoter and mammalian and insect cell transfection attempted with Lipofectamine LTX (Invitrogen; www.lifetechnologies.com) and X-tremeGENE (Roche; www.roche.com). Few positive cells (
Cell-penetrating peptide delivery of biologically active oct4 protein into cultured Takifugu rubripes spermary cells X. X. Yang*, X. N. Hou, B. Xu, X. Hao, G. J. Jiang and T. J. Fan Department of Marine Biology, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China
(Received 28 October 2013, Accepted 27 June 2014) Continuous cell culture of a puffer fish Takifugu rubripes has been established for efficient delivery of exogenous genes or proteins to cultured fish cells. Transcription factor oct4 was chosen for transduction into cultured fish cells because of its conserved structure and function between fish and mammals. In this work, the T. rubripes oct4 gene was cloned and expressed in Escherichia coli as a recombinant protein by introducing cell-penetrating peptide (CPP) poly-arginine (11R) and 6His-tag at the C-terminus. After purification, recombinant proteins were added to the growth medium and incubated with T. rubripes spermary cells. Recombinant proteins that crossed the cell membrane were detected in the cytoplasm and nucleus by western blot and immunofluorescent observation. The function of transduced oct4 as a transcription factor in fish cells was confirmed by driving green fluorescent protein expression in the pEGFP-1 reporter construct with the conserved specific oct4-binding sequence from mouse Mus musculus. Taken together, 11R can be an efficient CPP in delivering fusion proteins to cultured fish cells. © 2014 The Fisheries Society of the British Isles
Key words: 11R; cell culture; puffer fish; recombinant protein; transcription factor.
INTRODUCTION Fish cell cultures are of great importance for theoretical and practical fish biological and pathological research. Improvements of in vitro DNA transfection in fish cell lines can improve in vitro assays for heterologous fish cell gene expression (Hackett & Alvarez, 2000), advance fish DNA vaccination methods (Lorenzen et al., 2002; Sommerset et al., 2005; Kurath, 2008), allow the search for new promoters that are active in fish cells (Collet et al., 2004; Ruiz et al., 2008) and enhance studies of small interfering (si)RNA interference in permanently transformed fish cell lines (Ruiz et al., 2009). Fish cell transfection was first reported in the context of the calcium chloride method (Bearzotti et al., 1992), and this technique was later improved with commercially available liposomes such as Lipofectamine and Fugene 6 (Lopez et al., 2001; Ruiz et al., 2009). Generally, gene delivery efficiencies were low in fish cells compared to mammal cell cultures (Huang et al., 2011). *Author to whom correspondence should be addressed. Tel.: +86 532 82031793; email: [email protected]
1369 © 2014 The Fisheries Society of the British Isles
1370
X . X . YA N G E T A L.
For those genes that could be transduced successfully into fish cells, expression was restricted due to low promoter efficiency and varied transcriptional activity that depended on the cell type studied. Although human cytomegalovirus immediate early (CMV-IE) promoters can offer greater expression than promoters of most animal genes, these promoters are not equally functional in all cell types, especially in fish cells. Previously, Takifugu rubripes (Temminck & Schlegel 1850) oct4 was constructed into pcDNA3.1 under the control of the CMV-IE promoter and mammalian and insect cell transfection attempted with Lipofectamine LTX (Invitrogen; www.lifetechnologies.com) and X-tremeGENE (Roche; www.roche.com). Few positive cells (
