Biochimica et Biophysica Acta, © 1992

1114 (1992) 2 0 9 - 2 2 1

Elsevier Science Publishers B.V. All rights reserved

209 0304-419X/92/$05.00

B B A C A N 87257

Genes and functions: trapping and targeting in embryonic stem cells W e n d y S. Pascoe, R o l f K e m l e r and S t e p h e n A. W o o d Max-Pianck-b~stitutfiir bnmunbiologie, F,'eiburg(Germany) (Received 4 June

1992)

Contents I.

Introduction

!!.

Embryonic stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A, ES cell isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. M a i n t a i n i n g E ~ cells in undifferentiated state . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. in vitro differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

210 210

D. in r i v e d i f f e r e n t i a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

212

Gene targeting in ES cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Targeting selectable genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Targeting non-selectable genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Subtle mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Consecutive inactivation of both alleles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Phenotypic analysis of targeted mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

213 213

Enhancer trap and gene trap in ES cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enhancer trap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Gene Irap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7.17

Conclusion

219

111.

IV.

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A.

V.

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Acknowledgements References

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1. Introduction The number of genes isolated and cloned is presently quite large and modern molecular techniques ensure the daily addition of new sequences to the data banks. The elucidation of a gene's sequence permits its comparison with other genes and, based on the degree of homology, its placement into gene families. From these

Correspondence to: R. Kemler, Max-Planck-lnstitut fiir Immunbiologie, Stiibeweg 51, Postfaeh 1069, 7800 Freiburg, Germany.

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comparisons, clues to the possible function of the gene product may be gained. Emperically determining the functions of the gene products remains, however, one of the central challenges of biology. A myriad of in vitro techniques are available to help analyse function and not infrequently are the only feasible means through which to make progress. However, the ultimate test of function can only be performed in vivo. One of the major systems in which gene function is analysed in vivo is the mouse. There are many reasons underlying this choice. These include; the assumption that results from a mammalian system will be more

210 directly applicable to understanding human development and disease; mammalian cells are more easily cultured allowing results obtained from in vitro studies to be tested in vivo using the same species. The relatively short generation time and lower breeding costs of mice have seen it used as the 'typical' mammal. Historically, some insights into gene function have been provided by classical genetic studies in mice. These studies involve the identification of phenotypes arising from serendipitous, radiation-/chemically-induced or insertional mutations in the coding or control regions of genes. Extensive breeding segregation studies are then required to localise the affected gene to a particular chromosomal region. The availability of an increasing number of genetic markers, which subdivide the genome into everdecreasing fragments, has greatly facilitated the identification of mutated genes, At present. however, it is still a relatively time.consuming and labour-intensive process to go from phenotype to gene in the mouse. This is due primarily to the limitations of Mendelian-style analysis arising from the size of available mouse colonies, the generation time required and the inaccessibility of the developing mouse embryo. Classical genetic studies in lower organisms, especially Drosophila melanogaster and Caenorhabditis elegans, have, however, been more fruitful mainly because they do not suffer from the limitations listed above. These studies have identified a large number of genes involved in cell lineage and general body plan during development and have subsequently bee~ of enormous value ~o mammalian geneticists and developmental biologists. For example, the Hox genes, which are essential during mouse development, were initially isolated from the mouse genome on the basis of their sequence similarities to conserved sequence motifs within the homologous Drosophila genes. The major limitation of studies of gene function via random mutation is the lack of control over which particular genes are affected and the manner in which they arc affected. The isolation of genes as discrete units has, to some extent, overcome this barrier. One approach, which has been extensively utUised over the last decade, is the ectopie over-expression of genes in transt~enic animals, This approach has been particularly ~uccessful in mice and a number of important insights into the in rive function of genes have been gained, More recently, it has also become possible to investigate gene function via the precisely determined deletion or modification of genomic sequences, Using gene-targeting technology (homologous recombination between chromosomal and introduced DNA sequences) in pluripotent mouse embryonic stem (ES) cells a variety of mutations from complete deletion to highly-specific point mutations may be achieved, in this type of approach, the embryonic stem (ES) cell is an important tool, since these pluripotent embryo-derived

stem cells can be genetically manipulated in vitro and then reintroduced into the embryo where they participate fully in developmental processes including population of the germline. In this way, the genetic modification can be stably transmitted to future generations. The utility of the ES cell in the study of embryonic development does not end there, however. Recently, ES cells have played a central role in approaches designed to isolate and characterize new developmentally-regulated genes. We refer here to the enhancertrap and gene-trap studies, it is the purpose of this review to discuss the role of the ES cell in the functional analysis of previously cloned genes (homologous recombination) and as a tool for the isolation of new genes (enhancer and gene trap). II. Embryonic stem cells

Murine embryonic stem (ES) cells are pluripotcnt stem cell lines established directly from the inner cell mass of the 3.5-day blastocyst. They retain their pluripotency following culture and manipulation in vitro and upon reintroduction into the mouse blastocyst can contribute to all tissues, including the germline. The first successful isolation of ES cells was reported independently in 1981 by Evans and Kaufman [1] and Martin [2]. The isolation of ES cell lines was aided by pre-existing knowledge about the morphology, growth requirements and culture protocols for maintaining embryonal carcinoma cells (EC; derived from teratocarcinoma tumours) in an undifferentiated form. EC cells have, however, a number of experimental limitations [3]. One of the most significant being their limited ability to differentiate extensively both in culture and when introduced into a host embryo. Stem cells isolated directly from embryos have a number of advantages over EC cells: (1) ES cells can be derived from many mouse strains, including normal inbred strains, such as 129Sv [4-6] and C57BL/6 [7], and strains carrying defined mutations, tw~/t w,~ [8] and twls/twtx [9], or even from oocytes rendered parthenogenetic [ 10] or androgenic [ll]. (2) ES cell isolation is immediate and does not require a transforming event to facilitate growth in vitro. (3) Most ES cell lines stably retain euploid chromosome constitution. (4) ES cells can generate chimaeras with high efficiency when reintroduced into the embryo and contribute to all body tissues including the gcrmiinc.

11-,4. ES cell isolation Evans and Kaufman ovariectomized 129 strain mice on day 2.5 of pregnancy and applied a hormone treatment in order to delay blastocyst implantation. The rationale behind this protocol was that the delay would increase the number of ICM cells in each embryo and

2II thereby, enhance their chances to grow in vitro. Martin obtained immunosurgically [12] isolated ICM from fully-expanded blastocysts and cultured them in a medium conditioned by EC cells. The assumption here being that the factors secreted by the EC cells might aid the growth of the ES cells. Euploid, pluripotent ES cell lines have also been established from mouse blastocysts without the use of ovariectomy, immunosurgery and EC cell conditioned medium [13] and more recently, from disaggregated mouse morulae [14]. All of these techniques used a feeder layer - either permanent STO fibroblast lines or primary embryonic fibroblasts - to prevent the differentiation of the ES cells. As fibroblast-conditioned media alone is unable to prevent differentiation of the ES cells, this suggests that direct cell-cell contact may be necessary. It has been demonstrated, however, that pluripotent ES cell lines can be isolated in the absence of feeder cells in a culture medium supplemented with the regulatory factor Differentiation-Inhibiting Factor/Leukaemia-lnhibiting Factor ( D I A / L I F ) [15,161.

ll.B. Maintaining ES cells bl undifferentiated state In 1987, Smith and Hopper [17] demonstrated that medium conditioned by Buffalo Rat Liver (BRL) cells prevents the spontaneous differentiation of ES cells that occurs when they are grown in the absence of feeders. The effect was found to be fully reversible and the soluble peptide regulatory factor was named Differentiation Inhibiting Factor (DIA). DIA was subsequently found to be identical to both leukaemia inhibitory factor (LIF; a haematopoetic regulator which induces differentiation in M I myeloid leukaemic cells) and human interleukin fl~ DA cells (HILDA; supports proliferation of the routine interleukin-3-dependent leukaemic cell line DA-la)[18-20]. D I A / L I F has recently been shown to exist in two forms, matrix-associated (M) and diffusible (D), which are generated by alternate promoter usage [21]. The ability of feeders to suppress ES cell differentiation may therefore arise not only from the production of diffusible D I A / L I F but also due to the additional deposition of D I A / L I F in the extracellular matrix. Additional work by Rathjen and associates [22] has shown that the M form of D I A / L I F is present at low levels in undifferentiated ES cells and that the ES cells secrete a factor (possibly heparin binding growth factor; HBGF) which stimulates the expression of D I A / L I F (especially the D form) by fibroblasts. Interestingly, both the D- and M-forms have been shown to be induced in ES cells upon in vitro differentiation. This early activation of D I A / L I F expression during ES cell differentiation provides an autocrine regulatory mechanism whereby differentiation can proceed without exhaustion of the stem cell pool. Expression of D I A / L I F has also been

found in the uterine endometrial glands of the mouse on day four of pregnancy [23], in the extraembryonic region of the eggocylinder stage mouse embryo [22] and in the placenta of later stage mouse embryos [24], suggesting that D I A / L I F may play a paracrine regulatory role in differentiation during mammalian development.

II-C hH.~itro differentiation In the absence of feeder layers or DIA/LIF, ES cells have the capacity to differentiate into many cell types in vitro [4] either spontaneously or induced by chemical agents such as retinoic acid [25]. In suspension culture, ES cell colonies form embryoid bodies displaying endoderm, basal lamina, mesoderm and ectoderm cell types within 3 to 8 days. At this stage, they are morphologically similar to 6 to 8 day egg-cylinder stage embryos. From 8 to 10 days in culture, the embryoid bodies develop into large cystic structures and are considered analogous to the visceral yolk sac of the postimplantatio~; embryo on the basis ot their expression of alphafoetoprotein and transferrin. Cystic embryoid bodies are also capable of developing myocardium and blood islands [4] and when allowed to re-attach to an adhesive surface, they can give rise to nerve cells, contracting muscle cells, epithelial cells, melanocytes and chondrocytes [2]. Differentiation of ES cells in vitro may provide an unique system in which to examine the potential role o1" growth factors in early development at a time when the mouse embryo is the least accessible [26-28]. It is also possible that ES cells can be used to study a particular developmental process in vitro by altering the culture conditions to force the ES cells to differentiate down a particular pathway. Some attention has been focused in this regard to develop a system m which to study haematopoiesis. Recently, Wiles and Keller [29] demonstrated that when ES cells are cultured directly in semi-solid media (methyl cellulose) containing erythropoietin and interleukin-3, erythroeytes, macrophages, mast cells and neutrophils could be found within embryoid bodies. This work was supported by Burkert and associates [30] who demonstrated in crop bryoid bodies the presence of all colony-forming progenitor cells normally found in the haematopoietic organs of the developing mouse. Further, they described the temporal development of these cells and showed that the various progenitors reproducibly develop at specific stages during ES cell differentiation. ES cells have also been used to study switching of globin gene expression in vitro [31] and to correlate the expression of cytokines, their receptors and the /3globins with specific stages of haematopoietic development throughout ES cell differentiation [32]. Recently, a defined cultivation system has also been developed

212 for the differentiation of ES cells into spontaneously beating cardiomyocytes [33]. Such a system may have implications not only as a model in which to study cell differentiation but also as a model for pharmacological investigations of cardioactive drugs.

ISOLATIONOF [CMF R O M B~CYST

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ll-D. in-.t:a~'o differentiation As mentioned above, ES cells can colonise all tissues, including the germline, following their reintroduction into host blastocysts` Their utility as a bridge ~tween in-vitro and imvivo systems can be considered to be the most important feature of ES cells, Foreign DNA has bbeen demonstrated to be successfully intro. duced into ES cells by retroviral infection [5,34,35], calcium.phosphate precipitation [36,37] and electropo. ration [38,39]. These genetically-altered ES cell lines have been reported to be stable in culture with respect to morpholo~ aM karyotype and can be induced to differentiate normally to embryoid bodies under suitable culture conditions [~-42]. Their ability to con. tribute extensively to both somatic- and germ-cell-derived tissues is most commonly assessed by injection of transfected ES cells into host blastocysts [36,43] (Fig. I). The level of contribution from either host or ES cell component is dependent upon the initial proportion of ES/ICM cells (determined by the number of ES ceils injected) and the genotype of both the host and the ES cells. This strain-dependence is possibly a reflection of the relative rates of development of the donor and host. Germ-line-competent ES cell lines such as D3 [4,36], CCE [$] and ABI [6] have been derived from the 129 strain and colonise the germ-line when injected into blastoc~ts from some mouse strains, such as MFI [.S,43] and ~ T B L / 6 [44], although no comprehensive analysis of compatible donor/host combinations has ~ ¢ n reported. Recently, a germ-line-competent C~TBL/6 ES cell line has al~ been established [7] The sex of the ES cells and of the host blastocyst also plays a role in germ-line transmi~ion. Due to the instability of XX ES cells [45], most commonly used ES cell lines are XY and, therefore, become spermatozoa upon entering the germ-line. This may occur in a XY host or by 'sex conversion' of an XX host ~mbryo. Germ line transmission via the female is le.~,~c~mmon because of the dominant effect of the Y ch~rno~me [46]. Culture conditions and passage number of ES cells have also been suggested to be influential with p',spect to the ability of ES cells to give ri~ to germ-line chimaeras, It is apparent, however, that late passage number ES cells can efficiently colonise the germ line [47], Several criteria may be used here to assess the pluripotent state of cultured ES cell lines. (1) Maintenance of an ES phenotype in culture can be assessed using antibodies such as ECMAo7 [48] and SSEA-I [49] which are specific for undifferentiated ceils. Con-

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Fig. I. Transgenesis via embryonic stem coils. Embryonic stem (ES) cells are derived from the inner cell mass of normal or implantation° delayed hlastt~'ysts, They can be maintained in culture in a pluripotent state with a normal karyotype either on a fibroblastic feeder layer, in conditioned media or in the presence of DIA/LIF. Following manipulation and screening in vitro, they can be reintroduced into bh~sto~sts, from mice of a different coat colour, where they intermingle with host coils, Upon transfer to a pseudopregnant recipient, the U S cells participate in the normal development of the chimaeric embryo and contribute to all cell types, including the germ line. In the chimaeras donor ES cell and host cells develop in parallel but with no exchange of genetic material. Chimaeras are identified by coat colour, Mating chimaeric adults to mice of a coat coiner over which the ES cells are dominant, allows the rapid identification of transgenic offspring derived from the donor ES cells.

versely, the monoclonal antibody, TROMA-1 may be used to detect the contribution of differentiated cells in the culture [50] (~) Changes in karyotype are dependent on cuhure conditions and have also been observed to occur after transfection procedures, therefore, euploi0y shoal0 be confirmed by karyotype analysis. (3). Ability to differentiate extensively in in-vitro culture may provide an indication of ability to differentiate in rive. Despite careful culturing and rigourous testing of the ES cells, however, the ultimate test of their pluripotency is their ability to contribute to the development of a host embryo. That is, the maintenance of the correct phenotype in vitro is not a direct indication of pluripotency in rive. It has been suggested [44,46] that in most established ES cell lines, while the major-

213 ity of cells exhibit a typical ES morphology, only a fraction of these are truly totipotent and that germ-line transmission is the result of the ability of the embryo to discern normal from abnormal. Interestingly, ES cells have been isolated and maintained either on feeder layers or in LIF-supplemented medium and subsequently shown to contribute to the germ line. Although the extent of contribution from ES cells injected into blastocysts has been reported to be as high as 95% [6], several studies ha~e directly addressed the question of the developmental potential of ES cells when reintroduced into mouse embryos. In 1989, Beddington and Robertson [51] injected either single or multiple ES cells into host blastocysts and analysed their contribution to derivatives of the three primary tissue lineages (trophectoderm, primitive endoderm and epiblast) in midgestational conceptuses. It was coneluded that these ES cells most closely resemble early inner cell mass (ICM) cells in their developmental potential, since they were capable of colonizing trophectoderm and primitive endoderm derivatives at a low frequency, as well as producing a high rate of chimaerism in tissues of the foetus and extraembryonic mesoderm. Nagy and associates [52] compared the developmental potential of ES cells to day 3.5 ICM cells by aggregation with normal diploid embryos and developmentally compromised tetraploid embryos. When newborn ICM/tetraploid and ES/tetraploid chimaeric mice were examined they were found to be almost all exclusively ICM- or ES-derived; the tetraploid cells being found only in the yolk sac endoderm and trophectoderm lineage. These results suggested that in the mouse, ES cells alone are able to support development of the embryo proper. In another study, the fate of ES cells injected into blastocysts and morulae was followed using an ES cell line stably expressing lacZ under the control of an endogenous promoter [53]. This work demonstrated that injection of ES cells into morulae prior to ICM formation was an efficient way to increase the degree of chimaerism of the embryos suggesting that the precise stage at which embryos are injected might influence the efficiency of ES cell colonisation and subsequently the probability of obtaining germ line chimaeras. In both the tetraploid and morula experiments, however, an excessive contribution from the ES cells resulted in embryonic [53] or neonatal [52] lethality. This indicates that, while ES and ICM cells have many characteristics in common, they are not ultimately equivalent. In summary, ES cells are invaluable in their role as a bridge between in vitro and in vivo studies. They can be established from many mouse strains. In vitro, they are capable of differentiating into an amazing variety of cell types. When introduced into host embryos they can contribute to all tissues, including the germ line. Further, they can be stably transfected in vitro and

subsequently used to manipulate the germline, thereby providing a route to transgenesis. The next section of this review will address this last point; that is, deliberate modification of the genome by homologous recombination in ES cells. IlL Gene targeting in ES cells It was first reported in 1982 that mammalian somatic cells posses~ the enzymatic machinery for efficiently mediating ht~mologous recombination between newly-introduced non-replicating DNA molecules [54]. This finding subsequently stimulated a plethora of studies in the field we now refer to as 'gene targeting'. Early studies indicated that although the frequency of extrachromosomal homologous recombination was high [55,56], recombination between incoming DNA and cognate chromosomal regions occured largely at random. In fact, in this study, non-homologous integration was shown to occur at least 1000-times more frequently than homologous recombination [57,58]. Nonetheless, these experiments demonstrated the feasibility of altering, specifically, genes in cultured mammalian cells. Most cultured cell lines, however, contain at least two copies of a given gene, so that the inactivation of one allele is not likely to result in the loss of function. It became apparent, however, that the targeting of genes in ES cells would provide an avenue for the in-vivo analysis of the function of many genes. A three-step strategy was envisioned: (1) targeting of one gene copy in ES cells, (2) introduction of the manipulated ES cells into the germ line and (3) breeding of mice to homozygosity to create mutants.

111.,4. Targeting selectable genes A number of basic parameters which influence th~ frequency and integrity of homologous recombination have been investigated by targeting genes which are directly selectable in ES cells. In this regard, the targeting of the hypoxanthine-guanosine phosphoribosyl transferase (Hprt) gene has generally been the system of choice because of two primary features. Firstly, as the Hprt gene lies on the X chromosome, male ES cells are heterozygous for Hprt and only one copy of Hprt needs to be inactivated in order to yield a selectable phenotype. Secondly, Hprt is a doubly-selectable gene; that is, Hprt- mutants are resistant to the base analogue 6-thioguanine (6-TG) but fail to grow in hypoxanthine, aminopterin, thymidine.containing (HAT) medium. Early studies of Thomas and Capecchi [38] used the Hprt system to compare two classes of recombinant vectors: replacement (12) and insertion (O) (Fig. 2). They used a construct consisting of the neomycin phosphotransferase gene (neo), under the control of the

214 B. INSERTION

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Fig. 2, l~si~n of targeting vectors, (A) Replacement vectors, Repl~c~ment veeto~ are designed such that the regions of gene homology are ~eparat~d l~ the nonl~m~logous sequences of the selectable g~nc (SO) which ~places a e~Jing region, The vector is linearised at the fx)int of di~'ontinuity between the two regions of homology, Upon homologous recombination the vector, containing the replacemere of the ~.~t~lingregion with the selectable gene is substituted for the endogenous region, (B) Insertion vectors, Insertion vectors are linearised in adjacent regions of homology, Upon homologous recombination involving a double strand break the entire vector is inserted into the endogenous gene and results in partial duplication of the gene, Open areas represent introns: close hatched areas repre~en! exons; loose hatched areas represent sel~ctable genes (SG),

HSVotk promoter and polyoma enhancer, inserted into oxen 8 of a cloned fragment of the Hprt gene, In both v~ctors, the ¢leo 8ene was u,~d not only to disrupt the Hprt gene but also as a directly selectable marker, No d:i~rencc was observed between insertion and replacoment in terms of targeting frequency; one homologous recombination per 1000 random integrations was achieved, Because of the utility of replacement vectors in the positive-negative targeting regime for non-selectable genes (,~e below) and their comparative ease of construction this class of vector has been used most extensively to date, A recent rea~se~sment of replacement versus insertion vectors has produced contrasting results, however [$9], Again targeting the tlprt gene in E$ cells, Hasty and co-workers [59] found that insero t~on vectors target nine times more frequently than replacement vectors containing the ,same length of sequence homology, in addition, they noted that targeting with replacement vectors, in most instances, did not alter the locus in the predicted manner. When considering the combined effects of the fidelity and

rate of targeting, they conclude that insertion vectors are approx. 90-times more efficient. The weight of evidence from numerous targeting experiments of various genes indicates, however, that infidelity of targeting with replacement vectors is not a problem. The most comprehensive study to date analysed 13 replacement and 9 insertion vectors targeting two different sites in the HPRT gene [60]. The conclusion from these studies was that there is little difference in targeting efficiency and fidelity between the two classes of vectors. The only instance of targeting infidelity arose when a replacement vector derived from nonisogenic DNA was used. A ,dmilar replacement vector prepared from isogenic DNA targeted as efficiently as the corresponding insertion vector. At present, therefore, replacement vectors have two significant advantages over insertion vectors; (i) with insertion vectors, there is always the possibility of reversion to the wild-type locus and (ii) the ability to use the positive-negative strategy. The inability to use the positive-negative strategy with insertion vectors may be offset, however, by the use of a number of other strategies now available to enhance for homologous versus random integrations (see below). Increasing the length of sequence homology between vector and target gene has been reported to increase the frequency of targeting events over random integrations for both insertion and replacement vectors [38,68]. This study suggests that there is a minimum critical length of homology necessary to mediate homologous recombination and, at least for Hprt, this may bc shorter for insertion vectors [61]. Increases in homology from the minimal critical length (of around 1 kb for Hpn in ES cells) initially result in an almost exponential increase in targeting frequency until a plateau is reached (around 9 kb for Hprt) at which further increases in homology produce more modest increases in targeting frequency [38,61]. A less dramatic but still significant correlation between targeting frequency and increases in homology has also been observed in hybridoma cells [61]. In general, the size of nonhomologous regions are kept to a minimum a!though the presence of large regions of nonhomology outside regions of homology does not appear to affect targeting [63--65]. The fidelity of gene targeting in many instances has not been rigorously examined especially in experiments involving gene ablation. The advent, however, of experiments requiring the introduction of precise subtle mutations has prompted a reassessment of this parameter, Homologous recombination can be extremely precise, in one instance, only two single base pair deviations were detected in the analysis of a total of 80 kb from 44 independent ES cell clones containing a targeted Hprt gene [64]. The introduction of exogenous sequences by microinjection may result, however, in higher incidences of mutations [66,67].

215

Ill-B, Targeting non-selectable genes Unlike Hprt, most genes are present as two copies in the genome and are not directly selectable. Therefore, a number of strategies have been developed to increase the frequency of homologous recombination in relation to random integrations of the vector, in effect, these are mostly aimed at selecting against cells in which non-homologous events have occurred. The most widely used method to date is the positive/negative selection (PNS) procedure. This protocol uses a positive selection for cells that have incorporated the targeted vector into the genome and a negative selection step against the cells that have randomly integrated the vector. The vector used by Mansour and co-workers [63] for preliminary PNS experiments contained a neo gene inserted into an exon of the target gene and a herpes simplex thymidine kinase gene (HSV-tk) adjacent to the region of target homology. The neo gene has a dual action to disrupt the target gene and act as a selectable marker for cells which have integrated the vector; cells expressing the neo gene (neo +) will be neomycin (G418) resistant. The pivotal point of this approach is that the HSV.tk gene, located at the junction between homology and nonhomology, will be lost if the vector homologously recombines but will be retained in random integrations; HSV-tk- cells will be resistant to gancyclovir (GANC). Ultimately, cells correctly targeted for gene X will be X - n e o +HSVtk- or X - G418"GANC' and randomly-targeted cells will be X +neo +HSVtk + or X +G418rGANC '. Using this approach, the Hprt and int-2 genes were targeted [63] and a 2000-fold enrichment of targeted over random integration was reported for both genes. The absolute targeting frequency for hit-2, however, was 20-fold less than for Hprt. Because of its wide utility the PNS approach has been employed in the targeting of many genes in ES cells. The targeting of the Hox 1.3 [68], insulin-like growth factor Ii [69] and Wnt-1 [6] using PNS vectors has, however, only resulted in approximately !0-fold enrichment. In fact, in other studies enrichments as low as two-fold have been reported [70,71]. Further, the inclusion of tk cassettes at both ends of the targeting vector did not increase the enrichment factor [6,71]. Another positive-negative selection system used the diptheria toxin A-fragment as the negative selection marker and produced a 10-fold enrichment when the c-fyn locus was targeted [72]. A cautionary note is necessary at this point. The enrichment values given above were derived from comparisons within each particular study. As with targeting frequency, direct comparison of the enrichment factors obtained in the various studies are not possible due to differences in target loci and targeting procedures such as vector design, culture conditions and selection procedures.

The use of a promoterless positive selection marker has also been employed to select against randomly integrated background events. In this strategy the expression of the sclectable gene is dependent on the correct homologous recombination event where the gene is expressed as a fusion product or under the control of the transcriptional control elements of the targeted gene. This approach also results in a 10-100fold enrichment [40,68,73-76]. The disadvantage of this system is that the gene of interest must be expressed in ES cells. This restriction may not be so severe, however, since genes that have extremely low levels of expression have also been targeted in ES cells [68] and the frequency of targeting did not correlate with the level of expression in these studies [68,77]. It also appears that ES cells may be somewhat promiscuous in their expression patterns [68]. A variation on this approach has been reported by Le Mouellic and coworkers [78]. The lacZ marker gene was used to replace the Itox 3.1 gene, the lacZ being subsequently transcribed from the Hox 3.1 regulatory elements. The aim of this approach was not to aid the selection of successfully targeted cells but rather, as a way of tracing homologously recombined cells during development in vivo. A novel approach was adopted in an attempt to mutate the mouse homeobox gene, Hox 11 [79]. The targeting vector carrying a 20-bp mutation was introduced by microinjection, rather than the more usual electroporation. No selection was applied and homologous recombination events were selected by means of the l~oiymerase chain reaction (PCR). Correctly targeted events oco,~','d in 1 out of 150 microinjected ES cells; this was estimated to be a ratio of 1:30 homologous vs. non-homologous recombination. PCR was also used in a strategy to mutate the En-2 gene [80]. Similar to that suggested by Kim and Smithies [81], this strategy involved the integration of a neo-expressing/2-type vector and the subsc~uent use of PCR amiJlification. With this combination, one targeted event per 260 G418-resistant colonies was achieved.

III-C Subtle mutations Initial homologous recombination experiments were in effect gene ablation. In order to allow detailed study of gene structure and function or to mimic human genetic diseases resulting from small specific mutations, however, it is necessary to create subtle specific mutations which do not leave extraneous sequences in either the target or surrounding genes. Several studies have directly addressed this point. Two specific point mutations, located 20 bp apart, were introduced into the RNA polymerase gene in ES cells [82], The first mutation conferred resistance to the mushroom toxin a-amanatin (ama) and the second generated a restric-

216 tion fragment length polymorphism (RFLP) without alteration in the protein sequence. 1 in 30 successfully electroporated ES cells were found to be ama resistant and 3 0 - ~ % of these also carried the RFLP. Those clones carrying only the area gene were thought to have arisen through DNA mismatch repair rather than double crossover or gone conversion and so are probably an overestimation of the frequency of mutations arising from targeting events. A 4-bp mutation was inserted into the Hprt gene using an 'in-out' targeting procedure [83]. The 'in' step involved the introduction of the targeting vector carrying the 4-bp insertion within the region of target homolo~ into a Hprt- ES cell line (E14TG2a). Successfully targeted clones (Him +) were selected in HAT medium, The 'out' step required selection of HAT r clones in 6-TG medium for spontaneous revertants. ~ % of 6-TG r clones examined by Southern blot analysis were shown to have successfully excised the Hprt vector and 19 out of 20 of these retained the 4-bp insertion, The 'hit and run' procedure involves the introduction of a site-sI~cific mutation into a non-selectable gone by a two-step recombination process [84]. In addition to the mutation carried within sequences homologous to the target gone, the vector contains a nee gone and a HSV.tk gone. In the 'hit' step, targeting occurs by single reciprocal recombination generating a duplication of homology separated by the plasmid and the selection cassettes. Successfully targeted clones are selectable in (3418. In the 'run' step, single reciprocal intrachromosomal recombination (or 'reversion') removes nee, HSV.tk, the plasmid backbone and one copy of the duplicated homologous DNA, The loss of the HSVotk can be selee,'¢d for in FIAU, This procedure was used to create subtle mutations in both the Hprt and Hoe"2.6 genes with a high frequency, Studies by Reid and co-workers [85] show that the dual ~lvctability of the Hprt gone renders it an ideal marker for 'in-out" targeting procedures for nonse. lectable genes, That is, a subtle mutation can be created by electroporating the Hprt gone contained in a replacement (f~) vector into Hprt- ES cells and selecting in HAT medium. The surrounding Hprt sequences can then be removed by electroporating a . ~ n d f~-type construct containing the mutation but lacking Hprt .sequences, Successfully targeted cells can b¢ ~leeted in 6-TG medium, Alternatively, the co-electrol~ration of vector~ into ES ~ l h can allow the subtle modification of the gone of interest without the introduction of large stretches of non-homology into the same locus [86]. Although the frequency of double transfection was much lower than anticipated it may still prove a viable alternative in the next generation of subtle mutations in nonselectable genes. The generation of an insertional muta-

tion by the independent selectable marker cannot, however, be excluded.

III-D. Consecutive inactivation of both alleles The disruption of one allele may result in a dominant phenotype which precludes the generation of viable homozygous individuals. Therefore, several attempts have been made to consecutively inactivate both alleles of a target gene in ES ceils. The first of these, targeting of the pim-I gone [40], was achieved using the nee gone to disrupt the first allele and the hygromycin (tLvg) gone to disrupt the second. Expression of the marker genes was made dependent on the acquisition of transcriptional and translational initiation signals from the host DNA. Pim-I was thought to be a suitable locus for this approach, since it is highly expressed in ES cells. The a,2 subunit of the inhibitory hetorotrimeric guanine nucleotide-binding proteins is highly conserved in mammals and expressed in all cell types. Both alleles of the ai,, gone were inactivated using PNS; the first using nee and HSV.tk and the second using hyg and HSV-tk [41]. PNS was also used to test the requirement of keratin intermediate filaments for the formation and function of simple epithelium by sequential inactivation of both alleles of the mouse keratin 8 (inKS) gone in ES cells [42]. The first allele was inactivated using nee and HSV-tk and the second using the Hprt minigene [85]. in all of these experiments, ES cells containing wild-type, heterozygous or homozygous null-alleles were induced to differentiate in vitro. No phenotypic differences were observed between genotypes for any of the three loci, suggesting that none of them are required for ES cell differentiation in vitro. These cells do, however, present a null background into which mutations of the targeted genes can be introduced. Repeated rounds of gone targeting have also been used to consecutively modify one allele of the creatine kinase M (CKM) gone [87]. The CKM gone is known to be transcriptionally inactive in early embryogenesis. In this approach, a nee gone was used initially to disrupt the gone and replace the segment spanning the translation start site. Subsequently, a targeted enhancer replacement was achieved by insertion of the constitutively acting polyoma virus enhancer PyF441. This resulted in the functional activation of the silent CKM gone.

lll-E. Phenotypic analysis of targeted mutations The detailed analysis of the ever increasing number of mice homozygous for targeted genes is beyond the scope of this review. It is perhaps appropriate at this time, however, to attempt to discuss some of the direc-

217 tions this work may take in the foreseeable future. Homologous recombination in ES cells has resulted in the generation of mice of three phenotypic classes: (1) apparently normal phenotype, (2) abnormal phenotype in the hetero~,gous state, (3) abnormal phenotype in the homozygous state. No discernible phenotype was observed in either undifferentiated or differentiated ES cells following the targeting of both alleles of mouse keratin 8 [42], G-protein ai2 subunit [41] and pim-I [40]. This has led to the proposal that a number of genes, especially those of multigene families, are functionally redundant during development. This is further supported by the observation that even when some homozygous negative animals do present a phenotype the cell populations affected are often only a subpopulation of those normally expressing the gene. Mice heterozygous for the inactivation of the insulin-like growth factor 11 (IGF-II) are only 60% the size of normal litter mates [69]. This was the first example of an abnormal phenotype in the heterozygous state. A later study indicated, however, that IGF-Ii is subject to parental imprinting so that in effect the heterozygous mice have no functional IGF-II, since both copies have been inactivated by either gene targeting or imprinting [88]. A number of mutants have now been generated which display a phenotype when homozygous. These include the oncogenes c-myb [89], c.src [90], c-abl [91,92], Wnt-1 [6], the homeobox genes Hox 1.5 [93] and Hox 1.6 [94] and genes involved in haematopoiesis, such as /32-microglobulin [95,96] and the immunoglobulin/z-chain [97]. In immunoglobulin/z-chain null mutants, the complete spectrum of cell types which normally express the gene are absent [97]. The absence of ~2-microglobulin results in the failure of cytotoxic T cells to develop [95,96]. Although this protein is expressed in nearly all cells as part of the major histocompatability complex class i it has been postulated that it is only essential for the maturation of CD8 + cells. The results of the targeting experiments, therefore, support this hypothesis. In several other instances however, only a subpopulation of cells which normally express the gene are affected. Wnt-I is expressed on the dorsal midlirre along the entire length of the neursal tube but no phenotype is observed in wnt-1-/wnt-1- mice caudal to the cerebellum [6,98]. Similarly, Hox 1.5- and Hox 1.6-negative mice die at or shortly after birth, morphological analysis reveals that only a certain pol~ulation of cells develop abnormally [93,94]. A more dramatic example was observed in c-src and c-abl negative mice. Both these oncogenes are expressed in all tissues, however, only cells involved in bone (for c-src) [90] or lymphoid development (for c-abl) were affected in the homozygous mice [91,92]. An intermediate situation

was observed following the targeting of c-myb. In these mice, only the cell populations with the highest levels of expression are affected [89]. it seems, therefore, that following the disruption of both alleles of a gene, a phenotype ranging from apparently normal to total loss of the expressing cell populations can be expected. IV. Enhancer trap and gene trap in ES cells

The processes underlying tissue differentiation and organogenesis remain one of the central areas of investigation in developmental biology. Factors involved in these processes have yet to be elucidated, however, recent advances in molecular biological techniques have now made studies of this developmental time frame possible. In particular, two similar approaches, enhancer trap and gene trap, now provide the opportu. nity to isolate and characterize developmentally regulated genes. Both enhancer trap and gene trap strategies involve the introduction of reporter genes that lack all or part of the transcriptional control elements into target cells. The expression pattern of the reporter gene is, therefore, entirely dependent upon the cis.reg. ulating elements of the 'trapped' endogenous gene. Previous attempts to isolate developmentally regulated genes in early postimplantation embryo have been hampered by the inability to isolate pure cell populations and the inherent bias of RNA analysis sys.tems toward more highly expressed genes. The gene and enhancer trap approaches have an advantage over these systems because they are DNA traps and, the~cfore, have no such bias. In addition, reporter gene expression can be detected in individual cells. The assumptions in these DNA methods are that the reporter gene can be introduced randomly into all parts of the genome, *hat low levels of reporter gene expression can be detected and that this expression neither favours nor disadvantages the expressing cell. IV-A. Enhancer trap The enhancer trap was designed originally to identify and and functionally characterize cis regulatory elements by introduction of a known gene without enhancer [99-103]. When a reporter gene is placed 3' to a minimal promoter and the construct is used a:, a transgene, the enhancer trap technique becomes a useful method with which to identify new developmentally-regulated genes. The rationale behind the enhancer trap is that upon integration an endogenous enhancer will drive transcription from the minimal promoter of the reporter gene, thereby determining the pattern of reporter gene expression. This approach has been used in Drosophila and transgenic mice to reveal developmentally-regulated genes with unique

218 ENHANCER TRAP

temporal and spatial patterns of expression [104-106]. In all of these experiments iacZ, as the reporter gene, was coupled to a weak promoter. In Drosophila, 70% of 49 transformed fly lines showed a temporally and spatially regulated pattern of lac Z expression in embwos; many of these were neural specific patterns. In the mouse experiment [105], all of the 11 mice expressing lacZ, expressed it in a unique and distinct pattern. This indicated that expression in these cases is largely dependent on chromosomal position. The use of transgenic mouse lines to detect developmentally regulated genes is, however, limited by the number of integration events that can be analysed. A strate~ was therefore developed employing ES cells to ~men integration events and to clone the associated genes [107]+ ES cells can be transfected with enhancer trap constructs and selected in vitro for integration event~ Cell lines bearing stable integrations of the reporter construct can then ~ introduced into the blastococl cavity of host blastocysts and expression patterns can be analysed at various time-points in chimaerie embryos without the generation of transgenie mice (Fig, 3). Gossit~r and co-workers [107] used a construct in which the lacZ gene was fused in frame to a minimal promoter derived from the mouse heatshock protein 68 (hsp68) gene wh!ch provides a TATA box and a translation initiation codon but alone is not sufficient for expression of/~-galactosidase in ES cells. The construct a l ~ contained the bacterial SUiil + suppressor gene and a nee gene under the control of a HSV.tk promoter. From 60 independent G41B-resistant ES cell lines, 6 expre~,~ed lacZ in the undifferentiated state: that is, they stained blue with 5-bromo-

4-chloro-3-indolyi-B-D-galactopyranoside (X-GaD, (XGel is a chromot~enic substrate which produces a blue precipitate upon cleavage by/3-galactosidase). Of these, 70% were found to contain one to two copies of the intact lacZ gene, Two of these 'blue' clones (nee+/ Xoal +) and five 'white' clones (neo'/ Xgal~) were injected into blastocysts, 'White' clones were analysed in embryos because, although they do not express the lacZ 8ene in undifferentiated ES cells, the construct may have integrated into genes which are only active in differentiated cells, Of the clones analysed, one 'blue' and one 'white' line showed a spatially restricted pattern of expre~ion in day 9.5, day 10.5 and day 12.5 embryos, The 'blue' line was tested for germ-line transmi~,don, in transgenic embryos derived from one germ lin~ m~le, the lacZ ~xpre~ion pattern was identical to that ~ r v e d in chimaeric animals, The limitation of the enhancer trap is primarily that as some enhancers are located at distances greater than 10-15 kb from the promoter in either direction, cloning of the genomic sequences immediately surrounding the transgene will not necessarily identify the endogenous gene.

l++p mmmm 'P+I

neo

GENE TRAP

~ ~

I

el

n.

ELECTROPORATION G418SELECTION

+

X.GALSTAINING

+

"WHITE" CLONES aeo resistant laeZ nt~llive

"BLUE"CLONES aeo resisl~t

poaitlve

BLASTOCYSTINJECTION AND RiglMPLANTATION INTO PSEUDOPREGNANT RgClPIENT

+ X-GAL$TMNING AT STAGEOF INTEREST

+

NOr Ig.XPii~I~D AF'IIi~ D~IATION

I~gPBI£SSED IN SPATIALLYAND TEMPORALLY RESTRICTED PAI~I'~qN

CONSTh'UTIVIELY EXPRESSED

Fig, 3, Enhancer and 8cue trap in ES cells. Both enhancer and gene traps function on the same principle; that is, the pattern of expression of a reporter gene is determined by the cis-regulatory elements of a 'trapped' endogenous gene. The reporter gene shown here is I~cZ which codes for the bacterial /J-galactosidase gene. LacZ.ex. pre~sing cells can be easily identified, since the action of/~-galactosidase on the substrate X-gel produces a blue cleavage product, Both 'trapping' strategies aim employ a selectable marker (shown here as the neomycin gene, nee) to identity successfully transfected colonies, These selectable markers may be expressed from an internal constitutive promoter (,,hewn as the capital P) or as a fusion product with the reporter gene and hence also under the control of the trapped transcriptional control elements. The enhancer trap construct has a minimal promoter (shown as the small p) 5' to the reporter gene, The reporter gene in the gene trap construct has no transcriptional control elements, only a 5' splice accepter. Following electroporation into ES cells, succe~fully transfected cells are se. letted for the expression of the m'o gene by their resistance to G418. Duplicate colonies are generated and one is stained for/~-galacto. sidase activity. 'Blue" colonies indicate the integration of the 'trap' into an actively transcribed generate region. 'White' colonies may represent the location of the construct in a noncoding region (the majority of instances) or within a coding region which is silent in ES cells, Both 'blue' and 'white' cells can be reintroduced into host blastt~.'ysts and following transfer to pseudopregnant recipients contribute to normal development. Embryos may be either harvested in utero and stained with X-gal to determine the spatial pattern of expression or allowed to develop to term, in which case chimaeras may be identified by coat colour. Collection of embryos at different developmental time-points also gives a temporal dimension to the expression pattern.

219 IV-B. Gene trap

Gene (or promoter) trap differs from enhancer trap in that promoter sequences have also been deleted from the construct. Therefore, the reporter gene must integrate into an endogenous gene in frame and in the correct orientation in order to be expressed. Since a fusion gene product may not result in detectable galactosidase activity, a splice acceptor site is placed immediately 5' to the lacZ coding region. Vectors may even be constructed with triple splice acceptor sites to allow insertion into any reading frame. A gene trap construct will also contain a selectable gene such as neo under the control of a moderate to strong promoter. The frequency of successful integrations is obviously much lower with gene trap than with enhancer trap, however, ES cell culture allows the initial screening of large numbers of integration events in vitro. Successfully transfected clones are isolated by G418 selection and then analysed for lacZ expression. The in-vivo developmental expression of neo-resistant clones expressing lacZ is then analysed at various times during development in chimaeric mice generated by blastocyst injection of ES cell clones. Gossler and co-workers [107] used a gene-trap vector containing the lacZ gone, lacking a promoter and translation initiation signal, inserted in frame into the homeobox exon of the En-2 gene such that a splice acceptor is placed at the 5' end of lacZ. The construct also contained a neo gene under the control of the human /3-actin promoter. Of 600 G418-resistant colonies, 10 also expressed lacZ and 8 of these were established as ES cell lines. All 8 'blue' cell lines were tested by blastocyst injection; between day 9.5 and day 12.5 of development, three of these showed constitutive expression patterns, four others showed developmentally-regulated patterns and one failed to produce chimaeras. Another feature of the gene trap is that successful integration not only leads to expression of lacZ but may also create an insertional mutant. By breeding to homo~gosity, the function of recessive genes may be studied. Friedrich and Soriano [108] took advantage of this feature in their attempt to identify developmentally-regulated genes° They constructed a novel reporter gene encoding a fusion protein, ~-geo, with both /]-galactosidase and neomycin activities. DNA constructs including this gene were introduced into ES cells by electroporation or retroviral infection. Approx. 95% of G418-resistant colonies stained blue with XGal. These cell lines were then pooled prior to injection into blastocysts. Because approx. 15 different ES cell clones are injected per blastocyst, more than one transgenic line can be derived from a single chimaera, The transgenic offspring of germ-line-transmitting chimaeras were identified by Southern blot analysis of tail DNA. LacZ expression patterns were examined by

mating transgenic males with wild-type females and staining embryos at different stages of development. Staining patterns were spatially and temporally different between lines. To determine whether recessive phenotypes were associated with mutations in the 'trapped' genes, heterozygous mice from 24 strains were intercrossed and the genotype of the offspring was determined by Southern blot analysis of tail DNA. Homozygous offspring could not be isolated from nine strains, indicating that the insertions led to a recessive lethal phenotype. In the remaining 15 strains, homozygous animals appeared morphologically and behaviourally normal. This suggests that either (1) the genes trapped in these strains are functionally redundant during development, (2) insertion of the gene trap vector does not affect gene function or (3) the resulting phenotype is not detectable. V. Conclusion The combination of modern molecular techniques and ES cell culture has added a new dimension to studies of gene function and developmental biology. Homologous recombination in ES cells makes it possible to modify already identified genes in a predicted manner, while gene trap offers a new way to identify some of the genes involved in developmental processes. The basic parameters which determine the frequency and integrity of gene targeting have been established. This has led to the modification, ranging from total deletions to single-base-pair substitutions, of a number of genes. The resulting phenotypes have also ranged widely from not discernible to the absence of entire cell and tissue populations. These 'knock out' experiments o~ly represent an initial step in our understanding of the function of individual gene products, however, they do provide the platform for the next generation of experiments. The availability of null mutants now makes it possible to introduce and test the effect of a range of modified genes, as well as other physiological stimuli on a negative background. One example will be the exposure of mice lacking certain populations of the immune system to infectious agents which will facilitate our understanding of how the immune system functions. This new generation of homologous recombination-reintroduction experiments promises to be a very powerful tool in increasing our knowledge of the molecular processes underlying whole animal biology.

Acknowledgements The authors wish to thank Ernst.Martin Fiichtbauer, Lionel Larue, Klaus Schughart and Johan Wallin for critical comments on the manuscript. Wendy Pascoe is a fellow of the Alexander yon Humboldt Foundation

~0

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