- Email: [email protected]
The identification of new genes: Gene trapping in transgenic mice
The identification of new genes: gene trapping in transgenic mice William C Skarnes AFRC Centr.e for G e n o m e Research, Edinburgh, U K Ongoing efforts to clone, sequence and map genes in the mouse have far exceeded our ability to define their functional role. The generation of mutations is an important first step towards understanding the function of genes in normal mouse development and physiology. Gene trapping in embryonic stem cells provides an efficient method to identify, clone and mutate genes at random, permitting the functional analysis of new genes in mice. Current Opinion in Biotechnology 1993, 4:684-689
Introduction The potential to carry out a large genetic screen in the mouse has been made possible by the development of the embryonic stem (ES) cell system. ES cells can be grown in large numbers, screened in culture for desired genetic changes, and returned to the embryo where they can contribute to the germline. ES cells are n o w widely used for the disruption of cloned genes by homologous recombination en route to creating nmtant mice that lack or express an altered form of the targeted gene [1-3]. A second application has been to identify novel genes by introducing reporter constructs into ES cells and monitoring the expression of the reporter in ES cell-derived chimaeric embryos [4]. Enhancer-trap and gene-trapping vectors have been developed for this purpose and can detect developmentally regulated patterns of reporter gene expression at a high frequency [5]. Gene-trapping constructs are particularly useful because insertions of these vectors are expected both to create a mutation by disrupting the endogenous gene at the site of integration and also to facilitate the cloning of the mutated gene. The basic principle behind gene trapping is the use of reporter gene constructs that are activated following their integration within endogenous transcription units. Two types of gene-trapping vectors have b e e n develo p e d that differ in their requirements for reporter gene activation (see Fig. 1). Promoter-trap vectors simply consist of a reporter gene lacking a promoter; expression of the reporter requires insertion into an exon of a gene [6-8]. Gene-trap vectors contain a splice acceptor sequence upstream of a reporter [5,9-11]; integration of this type of vector within an intron of an endogenous gene is predicted to generate fusion transcripts through the use of the splice acceptor site. By cre-
ating a fusion transcript with the endogenous gene, promoter and gene-trap insertions are highly likely to be nmtagenic. Furthermore, as a portion of the interrupted endogenous gene is included in the fusion transcript, the mutated gene is immediately accessible to molecular cloning. Finally, insertions that activate reporter gene expression place the reporter gene under the control of the cis-acting regulatory sequences of the endogenous gene. Thus, the expression profile of the endogenous gene may be inferred by monitoring the expression of the reporter. By and large, the central predictions of gene trapping have n o w been confirmed [7,11,12%13°°]. In this review, I summarize the data demonstrating the feasibility of gene trapping and discuss the potential application of this technology as a genetic screen in the mouse.
Mechanism of gene trapping Promoter-trap versus gene-trap vectors A summary of the results obtained with various promoter-trap and gene-trap vectors is presented in Table 1. Gene-trap vectors are more efficient at trapping genes than promoter-trap vectors. This presumably reflects the fact that mammalian transcription units are composed chiefly of introns and, therefore, present a larger target for gene-trap vectors. It follows that gene-trap vectors may show a higher frequency of integration into larger transcription units composed of many exons, whereas promoter-trap insertions may yield a more representative sampling of the genome.
The molecular characterization of several promotertrap and gene-trap insertions has confirmed the predicted mechanisms of reporter gene activation. The
Abbreviations ES~embryonic stem; h/s---histidinal dehydrogenase gene; neo--neomycin phosphotransferase gene; RACE--rapid amplification of cDNA ends.
684
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e n d o g e n o u s genes associated with promoter-trap insertions are found in genomic DNA immediately flanking the site of insertion. Inverse PCR has b e e n used to clone sequences 5' to the insertion, and analysis of these sequence shows that these vectors target exons [13"°]. The e n d o g e n o u s exons associated with genetrap insertions have been cloned from cDNA generated from the fusion transcript using the 5' rapid amplification of cDNA ends (RACE) strategy [12°']. In all cases tested, the splice acceptor was used properly to generate a spliced fusion transcript.
Biases introduced by vector design Apart from the obvious requirement for the insertion of promoter-trap and gene-trap vectors into exons and introns of genes, respectively, other inherent biases have emerged from the molecular characterization of trapped genes. The reporter gene used, and the m o d e of delivering DNA into cells, a p p e a r to influence the site of vector integration.
Reporter gene The drug resistance genes neomycin phophotransferase (neo) and histidinol dehydrogenase (his) have
~- A(n)
Fig. 1. Hypothetical integration of genetrapping vectors that activate reporter gene (hatched) expression and disrupt the target gene. (a) The promoter trap vector requires insertions into an exon (light shading) of the target gene to generate a fusion transcript. In contrast, (b) the gene trap vector generates fusion transcripts through the use of a splice acceptor (SA) sequence following insertions into an intron of the target gene. In both cases, the resulting fusion transcripts are regulated by enhancer (E) and promoter (P) elements of the disrupted target gene and terminated at a polyadenylation signal (pA) supplied in the vector.
b e e n used as reporters in promoter-trap vectors and allow the direct recovery of trapped genes by selection in the appropriate drug [5,7]. Insertions isolated with these vectors occur upstream of the coding region of the e n d o g e n o u s gene, usually in the first untranslated exon [7,13°°]. The bias in favour of insertions in the 5' non-coding regions of genes presumably reflects a selection against the production of protein fusions that, in effect, c o m p r o m i s e the enzymatic activity of these reporters. The bacterial lacZ gene offers a more versatile reporter because its product, [~-galactosidase, can be easily detected in cells by histochemical staining with X-gal or other, more sensitive fluorescent derivatives. Furthermore, lacZ can also accommodate large amino-terminal fusions and still retain activity, so that insertions within coding regions of genes are accessible [11,13°°]. As positive drug selection does not exist for lacZ, however, it is necessary to include a promoterdriven selectable marker in the vector to recover cells that take up DNA. Consequently, only a small fraction of the cells that stably integrate these vectors contain the desired trap event. To circumvent this problem, the ~geo reporter has b e e n developed [12 °°] in which neo is fused, in frame, to the 3' end of [~-galactosidase. ~geo encodes the enzyme activities of both the lacZ and neo genes, and promises to be an ideal reporter for gene trapping because it combines the advantages of
685
686
Mammalian gene studies Table 1. Relative efficiency and target bias of promoter-trap and gene-trap vectors. Vector
Reporter gene
Mode of DNA transfection
Trapping frequency*
Detection frequency-{"
Expected target
Mutation frequency:[:
Reference
Promoter trap U3his
his
Retrovirus
10-4
I
U3neo
neo
Retrovirus
10 -4
1
NASTI
neo
5xl 0 -4
1
U31acZ
lacZ
Electroporation Retrovirus
pl~gal
lacZ
pGT4.5
lacZ
pSA[l]gal
lacZ
pSA[ggeo
~geo
MoMuLV ROSA[3gal ROSA[3geo
lacZ lacZ [3geo
4xl 0 -3
4xl 0 -3
Electropotation
10-3
10-3
Electropotation Electroporation Electroporation Retrovirus Retrovirus Retrovirus
2xi 0 -2
2xi 0 -2
5xi 0 -2
5x10 -2
5' Non-coding region 5' Non-coding region 5' Non-coding region 5' Non-coding or coding exons Any exon
N.D.
[6,13"']
2/4
[1 3 " ]
N.D.
[7]
N.D.
[8]
N.D.
[I I ]
Introns before coding exons Any introns
I/3
[I 2 " ]
I/3
[I I]
Gene trap
5x10 -2
1
Any introns
1/2
[11 ]
10-3 10-7 10-1
10-3 10 -1 1
5' Introns 5' Introns 5' Introns
N.D. N.D. 7/19
[9] [11 ] [11 ]
N.D. indicates analysis not done. *The number of trap events per theoretical number of cells that take up the vector. ?The number of trap events per number of cells remaining after selection. :[:Recessiveembryonic lethal mutations per total lines tested.
the lacZ reporter for creating protein fusions and the ability to monitor gene expression using drug selection.
Retrovirus infection versus DNA electroporation
Retroviral-based vectors offer a very efficient way to introduce DNA into cells. The number of insertions per cell may be controlled by varying the nmltiplici W of infection. Proviral DNA integrates as a single copy and causes little damage to surrounding cellular DNA, greatly simplifying the structure of insertion events. Fusion transcripts obtained with retroviral based vectors tend to contain a smaU contribution from the endogenous gene [11,13"']. This is consistent with other studies that have observed a preferential insertion of proviruses at the 5' end of genes [14-16]. In contrast, a much broader range of fusion transcript sizes is observed with a gene-trap vector introduced into cells by electroporation [12°']. Therefore, integration of naked DNA appears to occur by a different mechanism that may be more random than virus-mediated integration. Electroporation of cells is a less efficient means of introducing DNA into cells; only 0.001-0.1% of treated cells contain stable DNA insertions [17]. Despite this, the inefficiency of this method does not pose a problem for gene trapping in ES cells because of the large numbers of cells available for transfection. The mechanism by which electroporated cells take up DNA is not
well understood but has been suggested to involve the action of topoisomerse I [7]. The introduction of DNA vectors by electroporation can cause rearrangements in host DNA. To date, three insertions have shown no significant alterations in host DNA [7,18], whereas two others suffered ill-defined alterations in genomic DNA downstream from the insertion [19]. Electroporation can also yield complex arrangements of the vector. Tandem arrays of vector DNA are frequently observed at the site of integration [11,12°°]. With one gene-trap vector (pSAI3geo; [11]), the presence of concatomeric DNA results in the synthesis of multiple readthrough transcripts. In contrast, a similar vector design (pGT4.5) generates a single major fusion transcript, despite the' integration of tandem copies [12"]. It is not clear w h y these two vectors should behave differently and a more detailed analysis of pSAl3geo insertions is required. Deletions from the ends of the vector have also been observed that may, in some cases, compromise the effectiveness of a vector. For example, a splice acceptor sequence placed too close to the end of one gene-trap vector was frequently lost; consequently, the vector behaved as a promoter trap [19].
The reporter gene reflects endogenous gene activity The lacZ-based gene-trapping vectors permit the visualization of gene expression patterns during embryonic development. Histochemical staining of chimaeric or
Gene trapping in transgenic mice Skames 687 f
transgenic embryos shows a variety of lacZ expression patterns during embryogenesis [5,11,12"]. Studies with one vector design have demonstrated the good correlation between the distribution of endogenous transcripts and the pattern of l a c Z activity observed in embryos for two lines characterized to date [12"°]. Vectors based o n retrovirus have not been tested for their ability to report accurately endogenous gene activity. Proviral insertions occur within DNAse I hypersensitive sites in chromatin, regions that are likely to contain important regulatory sequences [14-16]. Interruption of these sequences may be likely to influence the expression of the target gene. For example, a proviral insertion into the collagen I gene has been shown to inappropriately repress the expression of this gene during embryonic development [20--22]. In addition, retroviral sequences possess intrinsic enhancer activity. These findings raise questions regarding the utility of retroviral vectors for reporting endogenous gene expression.
Trapping genes causes phenotypic abnormalities in mice The potential of gene trapping to create nmtations in genes has been confirmed in mice. Thus far, a total of 31 insertions have been transmitted to the germline, 13 (40 %) of which cause embryonic lethal phenotypes [11,12"°,13"°]. The absence of obvious phenotypes associated with many the of the trapped genes is in keeping with the lack of overt phenotypes seen with many genes targeted by homologous recombination, much to the chagrin of those involved. In general, the results in mice are consistent with genetic studies in other organisms, estimating that only one third of single-gene mutations have an observable effect [23,24]. Therefore, the identification of mutations by gene trapping also allows the recovery of mutations that otherwise would be inaccessible to classic genetics. Furthermore, in cases where an observable phenotype is present, l a c Z expression can mark the cells that express the mutated gene and greatly aid the phenotypic analysis of these mutations.
Tailoring gene-trap screens to find genes of interest Creating large numbers of random insertional nmtations in ES cells by gene trapping affords the opportunity for pre-selecting insertions of interest before continuing with the time-consuming and costly task of producing germline chimaeras and carrying out subsequent breeding to analyze phenotypes. Cell lines may be selected on the basis of one or more of the following criteria: structure of the disrupted gene, subcellular localization of the fusion product, reporter gene expression in chimaeric embryos, and expression in response to ES-cell differentiation or other stimuli in vitro.
Pre-screens based on gene structure The relative ease in cloning the trapped gene allows the DNA sequence to be the criteria for prescreening genes of interest. For example, putative regulators of cell-cell interactions, transcription factors, molecules involved in signal transduction, etc., may be identified by their structural similarity to k n o w n genes. Gene-trap vectors that generate protein fusions are preferable for this purpose because the coding region of the endogenous gene may be found in RACE-generated cDNA clones. Cloning and sequencing of genes trapped in ES cells also has wider implications for genome mapping efforts. The localization of [3-galactosidase activity to a variety of subcellular compartments has been documented in gene-trap cell lines [5,9,10,25"] and may provide clues regarding the structure of the trapped gene. Normally, [3-galactosidase is distributed throughout the cytoplasm. The localization of [3-galactosidase to other subcellular structures presumably reflects the acquisition of endogenous protein domains that act as sorting signals. For example, a gene-trap fusion to a zinc finger containing protein shows [3-galactosidase activity in the nucleus of cells [12"]. The potential of the gene-trap approach to detect insertions in genes encoding secreted or transmembrane proteins has also been explored (WC Skarnes, RSP Beddington, unpublished data). Expression of a [3-galactosidase fusion containing a signal sequence has been found to lack enzymatic activity. Therefore, previous gene-trap screens have missed insertions into this class of genes. By including a transmembrane domain upstream of [3galactosidase in a gene-trap vector, enzymatic activity is restored in fusions with genes that encode aminoterminal signal sequences. Thus, insertions within cellsurface molecules are preferentially detected. Potential modifications in gene-trap vector designs may be envisioned to capture other classes of genes.
Pre-screens based on patterns of reporter gene expression Monitoring reporter gene activity in chimaeric embryos may be useful to find candidate genes relevant to a particular embryonic stage, developmental process, or cell lineage. Recently, 279 independent gene-trap insertions have been screened for l a c Z expression in chimaeric 8.5 day old embryos [25"]; 13 % of the cell lines showed restricted sites of expression, 32 % showed expression in most or all cells, and the remainder showed no expression. At least a third of the lines that showed no expression at 8.5 days were found to express l a c Z at later stages. Therefore, roughly two thirds of the genes expressed in ES cells display developmentally regulated patterns of expression in embryos. Simplified techniques for generating highly chimaeric embryos by morulae aggregation have b e e n developed [26"], which should increase the number of ES cell lines that can be analyzed in this way; however, a pre-screen based on expression patterns remains a significant undertaking. The differentiation of ES cell lines in vitro may provide
688
Mammalian gene studies an alternative method for identifying genes expressed in particular cell lineages. When grown in suspension, blastocyst-derived ES cells spontaneously give rise to a number of identifiable cell types including cardiac muscle, red blood cells, neurons, yolk-sac endoderm, and vascular endothelial cells [27,28]. Furthermore, defined culture conditions using various combinations of growth factors, have been established for the efficient and reproducible recovery of specific haematopoetic cell types [29]. A correlation of reporter gene expression with the appearance of specific cell types may help to pinpoint genes important in the differentiation of desired cell lineages.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest o, of outstanding interest 1.
CAPECCHIMR: Altering the G e n o m e by Homologous Recombination. Science 1989, 244:1288-1292.
2.
ROSSANTJ: Gene Disruption in Mammals. Curt Opin Genet Dev 1991, 1:236--240.
3.
JOYNERAL: Gone Targeting and Gene Trap Screens using Embryonic Stem Cells: New Approaches to Mammalian Development. Bioessays 1991, 13:649-656.
4.
SKARNESWC: Entrapment Vectors: A N e w Tool for Mammalian Genetics. Biotechnology 1990, 8:827-831.
5.
GOSSLERA, JOYNER AL, ROSSANTJ, SKARNESw e : Mouse Embryonic Stem Cells and Reporter Constructs to Detect Developmentally Regulated Genes. Science 1989, 244:463--465.
6.
VON MELCHNER H, REDDY S, RULEY HE: Isolation of Cellular Promoters by Using a Retrovirus Promoter Trap. Proc Natl Acad Sci USA 1990, 87:3733-3737.
7.
MACLEODD, LOVELL--BADGER, JONES S, .JACKSON I: A Prom o t e r Trap in Embryonic Stem (ES) Cells Selects for Integration of DNA into CpG Islands. Nucleic Acids Res 1991, 19:17-23.
8.
REDDYS, DEGREGOR!JV, VON MELCHNERH, RULEYHE: Retrovirus Promoter-Trap Vector to Induce lacZ Gene Fusions in Mammalian Cells. J Virol 1991, 65:1507-1515.
9.
BRENNERDG, L1N-CHAO S, COHEN SN: Analysis of Mammalian Cell Genetic Regulation in Situ by Using Retrovirus-Derived "Portable Exons ~ Carrying the Escherichia coil lacZ Gene. Proc Natl Acad Set USA 1989, 86:5517-5521.
10.
KERR WG, NOLAN GP, SERAFIN1 AT, HERZENBERG LA: Transcriptionally Defective Retroviruses Containing lacZ for t h e in Situ Detection of E n d o g e n o u s Genes and Developmentally Regulated Chromatin. Cold Spring Harb Syrup Quant Biol 1989, 54:767-776.
Conclusions
11.
In the past two years, the accumulated molecular and genetic data has validated the feasibility of gene trapping in ES cells to identify and mutate genes in the mouse. Thousands of gene-trap mutations can be conveniently obtained in culture a n d - - g i v e n the current rate of gemfline transmission and a reasonable amount of s p a c e - - i n the order of a hundred mutant mouse strains could be generated and analyzed by one laboratory over a period of two to three years. The rate of producing mutant mice by gene trapping is one to two orders of magnitude greater than that by targeted mutagenesis. Moreover, the development of prescreening strategies in ES cells should provide a versatile way to tailor screens towards specific interests.
FRIEDRICHG, SORIANOP: Promoter Traps in Embryonic Stem Cells: A Genetic Screen to Identify and Mutate Developmental Genes in Mice. Genes Dev 1991, 5:1513-1523.
SKARNESWC, AUERBACHA, JOYNER AL: A Gene Trap Approach in Mouse Embryonic Stem Cells: The lacZ Reporter is Activated by Splicing, Reflects Endogenous Gene Expression, and is Mutagenic in Mice. Genes Dev 1992, 6:903-918. Confirms all of the predictions o f the gene-trapping strategy. Using 5' RACE, the e n d o g e n o u s g e n e associated with three lacZ-based genetrap insertions is c k m e d and the proper use of the splice acceptor site is demonstrated. For two insertions, the pattern of lacZ expression in embryos is ~shown to match the normal distribution of e n d o g e n o u s transcripts. T w o of the three insertions tested cause phenotypic abnormalities in mice. O n e of these is an insertion into a novel g e n e expressed widely during development that causes peri-natal death in h o m o z y g o u s animals. The other is an insertion into a zinc-finger gene expressed in neural cells that results in mild growth retardation after birth.
Recently, one group has carried out a prescreen of gene-trap cell lines that are activated or repressed u p o n treatment with retinoic acid (L Forrester, W Wurst, personal communication). The effects of retinoic acid on axial patterning and limb development are well known, and genes responsive to retinoic acid nmy play a role in these processes. In a screen of 200 genetrap cell lines that expressed lacZ after treatment with retinoic acid for two days, expression was induced in nine cell lines and repressed in 11 cell lines. Many of these lines (70 %) showed spatially restricted expression in regions of the embryo known to be influenced by retinoic acid, that is, in the developing nervous system, limb buds and heart. Similar pre-screens, using a variety of other factors or morphogens, represent an exciting application of the gene-trap approach.
Acknowledgements I wish to thank L Forrester and W Wurst for communicating unpublished data, a n d L Forrester a n d A Smith for helpful c o m m e n t s and discussions. WC Skarnes is supported by an HFSP long-term fellowship.
12. e.
13. o•
VON MELCHNER H, DEGREGORI JV, RAYBURN H, REDDY S, FRIEDEL C, RULEY HE: Selective Disruption of Genes Expressed in Totipotent Embryonal Stem Cells. Genes Dev 1992, 6:919-927. Sequences upstream of nine retroviral promoter-trap insertions are cloned using inverse PCR. Flanking probes from five ES cell lines detected transcripts, and one clone is identified as the REX-1 transcription factor. T w o of four lines transmitted to the germline cause embryonic-lethM phenotypes. 14.
SHERIDEN U, RHODES K, BREINDI, M: Transcriptionally Active G e n o m e Regions are Preferred Targets for Retroviral Integration. Mol Cell Biol 1990, 64:907-912.
Gene trapping in transgenic m i c e Skarnes 15.
VIJAYAS, STEFFEN DL, ROBINSON HL: Acceptor Sites for Retro-
16.
ROHDEWOLDH, WEIHER H, REIK W, JAENISCH R, BREINDL H: Retrovirus Integration and Chromatin Structure: Moloney Murine Leukemia Proviral Integration Sites Map Near DNAse I Hypersensitive Sites. J Virol 1987, 61:336-343.
24.
viral Integrations Map Near DNase I-Hypersensitive Sites in Chromatin. J F~rol 1986, 60:683--692.
17.
BOGGS SS, GREGG RG, BORENSTEIN N, SMITHIES O: Efficient Transformation and Frequent Single Site, Single Copy Insertion of DNA can be Obtained in Mouse Erytholeukemia Cells Transformed by Electroporation. Exp Hematol 1986, 149:988--994.
18.
SOININENR, SCHOOR M, HENSELING U, TEPE C, KISTERS--WOIKE B, ROSSANTJ, GOSSLERA: The Mouse Enhancer Trap Locus 1 (Etl-1): A Novel Mammalian Gene Related to Drosophila and Yeast Transcriptional Regulator Genes. Mech Dev 1993, 39:111-123.
19.
NIWA H, ARAKI K, KIMURA S, TAN1GUCHI S, WAKASUG1 S, YAMAMUI~AK: An Efficient Gene-Trap Method using Poly A Trap Vectors and Characterization of Gene-Trap Events. J Btochem 1993, 113:343-349.
20.
BREINDL MK, HARBERS K, JAENISCH R: Retrovirus-Induced Lethal Mutation in Collagen I Gene of Mice is Associated with Altered Chromatin Structure. Cell 1984, 38:9-16.
21.
JAHNERD, JAENISCH R: Retrovirus-Induced de Novo Methylation of Flanking Host Sequences Correlates with Gene Inactivity. Nature 1985, 315:594-597.
22.
KRATOCHWILK, VON DER MARK K, KOLLAR E, JAENISCH R, MOOSLEHNER K, SCHWARTZM, MASSE K, GMACHL I, HARBERS K: Retroviral-Induced Mutation in Movl3 Mice Affects Collagen I Expression in a Tissue-Specific Manner. Cell 1989, 57:807-816.
23.
GOEBLMG, PETES TD: Most of the Yeast Genomic Sequences are Not Essential for Cell Growth and Division. Cell 1986, 46:983-922.
WILSONC, KURTH R, BELLEN MJ, O'KANE CJ, GROSSNIKLAUS U, GEHRING WJ: P-Element-Mediated Enhancer Detection: An Efficient Method for Isolating and Characterizing Developmentally Regulated Genes in Drosophila. Genes Dev 1989, 3:1301-1313.
25. •
WURSTW, ROSSANTJ, PRIDEAUX V, KOWNACKAM, JOYNER A, HILL D, GUILLEMOT F, GASCA S, AUERBACH A, ANG S-L: An Embryonic Expression Screen for Spatially Restricted Genes using Gcne Trap Integrations in ES Cell Chimaeras. Mech Dev 1993, in press. A screen o f 279 gene-trap cell lines for expression in 8.5 day embryos reveals developmentally regulated patterns in two thirds of the cases. 26. -
WOOD SA, ALLENND, ROSSANTJ, AUERBACHA, NAGY A: NonInjection Methods for the Production of Embryonic Stem Cell-Embryo Chimaeras. Nature 1993, 365:87-89. Describes time-saving m e t h o d s for generating ES cell derived chimaeras by morulae agg~regation. These can be used to more easily carry out a prescreen based on embryonic expression of the reporter.
27.
DOETSCHMANTC, EISTE+ITER H, KATZ M, SCHMIDT W, KELLER R: The in Vitro Development of Blastocyst-Derived Embryonic Stem Cell Lines: Formation of Visceral Yolk Sac, Blood Islands and Myocardium. J Emb~ol Exp Morph 1985, 87:27~i5.
28.
RISAUW, SARIOLAH, ZERWES MG, SASSEJ, EKBLOM P, KEMLER R, DOETSCHMAN T: Vasculogenesis and Anglogenesis in Embryonic-Stem-Cell-Derived Embryoid Bodies. Development 1988, 102:471-478.
29.
WILES MV, KELLER G: Multiple Hematopoetic Lineages Develop from Embryonic Stem (ES) Cells in Culture. Development 1991, 111:259-267.
WC Skarnes, AFRC Centre for G e n o m e Research, King's Buildings, West Mains Road, Edinburgh EH9 3JQ, UK.
689
Introduction The potential to carry out a large genetic screen in the mouse has been made possible by the development of the embryonic stem (ES) cell system. ES cells can be grown in large numbers, screened in culture for desired genetic changes, and returned to the embryo where they can contribute to the germline. ES cells are n o w widely used for the disruption of cloned genes by homologous recombination en route to creating nmtant mice that lack or express an altered form of the targeted gene [1-3]. A second application has been to identify novel genes by introducing reporter constructs into ES cells and monitoring the expression of the reporter in ES cell-derived chimaeric embryos [4]. Enhancer-trap and gene-trapping vectors have been developed for this purpose and can detect developmentally regulated patterns of reporter gene expression at a high frequency [5]. Gene-trapping constructs are particularly useful because insertions of these vectors are expected both to create a mutation by disrupting the endogenous gene at the site of integration and also to facilitate the cloning of the mutated gene. The basic principle behind gene trapping is the use of reporter gene constructs that are activated following their integration within endogenous transcription units. Two types of gene-trapping vectors have b e e n develo p e d that differ in their requirements for reporter gene activation (see Fig. 1). Promoter-trap vectors simply consist of a reporter gene lacking a promoter; expression of the reporter requires insertion into an exon of a gene [6-8]. Gene-trap vectors contain a splice acceptor sequence upstream of a reporter [5,9-11]; integration of this type of vector within an intron of an endogenous gene is predicted to generate fusion transcripts through the use of the splice acceptor site. By cre-
ating a fusion transcript with the endogenous gene, promoter and gene-trap insertions are highly likely to be nmtagenic. Furthermore, as a portion of the interrupted endogenous gene is included in the fusion transcript, the mutated gene is immediately accessible to molecular cloning. Finally, insertions that activate reporter gene expression place the reporter gene under the control of the cis-acting regulatory sequences of the endogenous gene. Thus, the expression profile of the endogenous gene may be inferred by monitoring the expression of the reporter. By and large, the central predictions of gene trapping have n o w been confirmed [7,11,12%13°°]. In this review, I summarize the data demonstrating the feasibility of gene trapping and discuss the potential application of this technology as a genetic screen in the mouse.
Mechanism of gene trapping Promoter-trap versus gene-trap vectors A summary of the results obtained with various promoter-trap and gene-trap vectors is presented in Table 1. Gene-trap vectors are more efficient at trapping genes than promoter-trap vectors. This presumably reflects the fact that mammalian transcription units are composed chiefly of introns and, therefore, present a larger target for gene-trap vectors. It follows that gene-trap vectors may show a higher frequency of integration into larger transcription units composed of many exons, whereas promoter-trap insertions may yield a more representative sampling of the genome.
The molecular characterization of several promotertrap and gene-trap insertions has confirmed the predicted mechanisms of reporter gene activation. The
Abbreviations ES~embryonic stem; h/s---histidinal dehydrogenase gene; neo--neomycin phosphotransferase gene; RACE--rapid amplification of cDNA ends.
684
© Current Biology Ltd ISSN 0958-1669
Gene trapping in transgenic mice Skarnes (a)
ATG
pA
Promoter trap vector --4// / . A I Reporter T
Target gene ~
+
GI
[]
P RNA transcript i ~
Promoter
trap ( insertion -
--
~
m\
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~
p RNA
,
~'
pA~/,
~},
/ / 4
~l---J
>2Reporter ~ 2
transcript (b)
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~ A(nl
SA pA Oene trap vector ~ / / , A I Reporter Target [~g_~ + gene ~ ) ~ AI~I [ ] .....
I
ii,,I [ - pA
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P RNA transcript I % Gene trap Insertion ( ~ A T G
*~
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, ~'"
pA ~>-~/// ~s~'l SA Reporter /
© 1993CurrentODmonmBiotechnology
e n d o g e n o u s genes associated with promoter-trap insertions are found in genomic DNA immediately flanking the site of insertion. Inverse PCR has b e e n used to clone sequences 5' to the insertion, and analysis of these sequence shows that these vectors target exons [13"°]. The e n d o g e n o u s exons associated with genetrap insertions have been cloned from cDNA generated from the fusion transcript using the 5' rapid amplification of cDNA ends (RACE) strategy [12°']. In all cases tested, the splice acceptor was used properly to generate a spliced fusion transcript.
Biases introduced by vector design Apart from the obvious requirement for the insertion of promoter-trap and gene-trap vectors into exons and introns of genes, respectively, other inherent biases have emerged from the molecular characterization of trapped genes. The reporter gene used, and the m o d e of delivering DNA into cells, a p p e a r to influence the site of vector integration.
Reporter gene The drug resistance genes neomycin phophotransferase (neo) and histidinol dehydrogenase (his) have
~- A(n)
Fig. 1. Hypothetical integration of genetrapping vectors that activate reporter gene (hatched) expression and disrupt the target gene. (a) The promoter trap vector requires insertions into an exon (light shading) of the target gene to generate a fusion transcript. In contrast, (b) the gene trap vector generates fusion transcripts through the use of a splice acceptor (SA) sequence following insertions into an intron of the target gene. In both cases, the resulting fusion transcripts are regulated by enhancer (E) and promoter (P) elements of the disrupted target gene and terminated at a polyadenylation signal (pA) supplied in the vector.
b e e n used as reporters in promoter-trap vectors and allow the direct recovery of trapped genes by selection in the appropriate drug [5,7]. Insertions isolated with these vectors occur upstream of the coding region of the e n d o g e n o u s gene, usually in the first untranslated exon [7,13°°]. The bias in favour of insertions in the 5' non-coding regions of genes presumably reflects a selection against the production of protein fusions that, in effect, c o m p r o m i s e the enzymatic activity of these reporters. The bacterial lacZ gene offers a more versatile reporter because its product, [~-galactosidase, can be easily detected in cells by histochemical staining with X-gal or other, more sensitive fluorescent derivatives. Furthermore, lacZ can also accommodate large amino-terminal fusions and still retain activity, so that insertions within coding regions of genes are accessible [11,13°°]. As positive drug selection does not exist for lacZ, however, it is necessary to include a promoterdriven selectable marker in the vector to recover cells that take up DNA. Consequently, only a small fraction of the cells that stably integrate these vectors contain the desired trap event. To circumvent this problem, the ~geo reporter has b e e n developed [12 °°] in which neo is fused, in frame, to the 3' end of [~-galactosidase. ~geo encodes the enzyme activities of both the lacZ and neo genes, and promises to be an ideal reporter for gene trapping because it combines the advantages of
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Mammalian gene studies Table 1. Relative efficiency and target bias of promoter-trap and gene-trap vectors. Vector
Reporter gene
Mode of DNA transfection
Trapping frequency*
Detection frequency-{"
Expected target
Mutation frequency:[:
Reference
Promoter trap U3his
his
Retrovirus
10-4
I
U3neo
neo
Retrovirus
10 -4
1
NASTI
neo
5xl 0 -4
1
U31acZ
lacZ
Electroporation Retrovirus
pl~gal
lacZ
pGT4.5
lacZ
pSA[l]gal
lacZ
pSA[ggeo
~geo
MoMuLV ROSA[3gal ROSA[3geo
lacZ lacZ [3geo
4xl 0 -3
4xl 0 -3
Electropotation
10-3
10-3
Electropotation Electroporation Electroporation Retrovirus Retrovirus Retrovirus
2xi 0 -2
2xi 0 -2
5xi 0 -2
5x10 -2
5' Non-coding region 5' Non-coding region 5' Non-coding region 5' Non-coding or coding exons Any exon
N.D.
[6,13"']
2/4
[1 3 " ]
N.D.
[7]
N.D.
[8]
N.D.
[I I ]
Introns before coding exons Any introns
I/3
[I 2 " ]
I/3
[I I]
Gene trap
5x10 -2
1
Any introns
1/2
[11 ]
10-3 10-7 10-1
10-3 10 -1 1
5' Introns 5' Introns 5' Introns
N.D. N.D. 7/19
[9] [11 ] [11 ]
N.D. indicates analysis not done. *The number of trap events per theoretical number of cells that take up the vector. ?The number of trap events per number of cells remaining after selection. :[:Recessiveembryonic lethal mutations per total lines tested.
the lacZ reporter for creating protein fusions and the ability to monitor gene expression using drug selection.
Retrovirus infection versus DNA electroporation
Retroviral-based vectors offer a very efficient way to introduce DNA into cells. The number of insertions per cell may be controlled by varying the nmltiplici W of infection. Proviral DNA integrates as a single copy and causes little damage to surrounding cellular DNA, greatly simplifying the structure of insertion events. Fusion transcripts obtained with retroviral based vectors tend to contain a smaU contribution from the endogenous gene [11,13"']. This is consistent with other studies that have observed a preferential insertion of proviruses at the 5' end of genes [14-16]. In contrast, a much broader range of fusion transcript sizes is observed with a gene-trap vector introduced into cells by electroporation [12°']. Therefore, integration of naked DNA appears to occur by a different mechanism that may be more random than virus-mediated integration. Electroporation of cells is a less efficient means of introducing DNA into cells; only 0.001-0.1% of treated cells contain stable DNA insertions [17]. Despite this, the inefficiency of this method does not pose a problem for gene trapping in ES cells because of the large numbers of cells available for transfection. The mechanism by which electroporated cells take up DNA is not
well understood but has been suggested to involve the action of topoisomerse I [7]. The introduction of DNA vectors by electroporation can cause rearrangements in host DNA. To date, three insertions have shown no significant alterations in host DNA [7,18], whereas two others suffered ill-defined alterations in genomic DNA downstream from the insertion [19]. Electroporation can also yield complex arrangements of the vector. Tandem arrays of vector DNA are frequently observed at the site of integration [11,12°°]. With one gene-trap vector (pSAI3geo; [11]), the presence of concatomeric DNA results in the synthesis of multiple readthrough transcripts. In contrast, a similar vector design (pGT4.5) generates a single major fusion transcript, despite the' integration of tandem copies [12"]. It is not clear w h y these two vectors should behave differently and a more detailed analysis of pSAl3geo insertions is required. Deletions from the ends of the vector have also been observed that may, in some cases, compromise the effectiveness of a vector. For example, a splice acceptor sequence placed too close to the end of one gene-trap vector was frequently lost; consequently, the vector behaved as a promoter trap [19].
The reporter gene reflects endogenous gene activity The lacZ-based gene-trapping vectors permit the visualization of gene expression patterns during embryonic development. Histochemical staining of chimaeric or
Gene trapping in transgenic mice Skames 687 f
transgenic embryos shows a variety of lacZ expression patterns during embryogenesis [5,11,12"]. Studies with one vector design have demonstrated the good correlation between the distribution of endogenous transcripts and the pattern of l a c Z activity observed in embryos for two lines characterized to date [12"°]. Vectors based o n retrovirus have not been tested for their ability to report accurately endogenous gene activity. Proviral insertions occur within DNAse I hypersensitive sites in chromatin, regions that are likely to contain important regulatory sequences [14-16]. Interruption of these sequences may be likely to influence the expression of the target gene. For example, a proviral insertion into the collagen I gene has been shown to inappropriately repress the expression of this gene during embryonic development [20--22]. In addition, retroviral sequences possess intrinsic enhancer activity. These findings raise questions regarding the utility of retroviral vectors for reporting endogenous gene expression.
Trapping genes causes phenotypic abnormalities in mice The potential of gene trapping to create nmtations in genes has been confirmed in mice. Thus far, a total of 31 insertions have been transmitted to the germline, 13 (40 %) of which cause embryonic lethal phenotypes [11,12"°,13"°]. The absence of obvious phenotypes associated with many the of the trapped genes is in keeping with the lack of overt phenotypes seen with many genes targeted by homologous recombination, much to the chagrin of those involved. In general, the results in mice are consistent with genetic studies in other organisms, estimating that only one third of single-gene mutations have an observable effect [23,24]. Therefore, the identification of mutations by gene trapping also allows the recovery of mutations that otherwise would be inaccessible to classic genetics. Furthermore, in cases where an observable phenotype is present, l a c Z expression can mark the cells that express the mutated gene and greatly aid the phenotypic analysis of these mutations.
Tailoring gene-trap screens to find genes of interest Creating large numbers of random insertional nmtations in ES cells by gene trapping affords the opportunity for pre-selecting insertions of interest before continuing with the time-consuming and costly task of producing germline chimaeras and carrying out subsequent breeding to analyze phenotypes. Cell lines may be selected on the basis of one or more of the following criteria: structure of the disrupted gene, subcellular localization of the fusion product, reporter gene expression in chimaeric embryos, and expression in response to ES-cell differentiation or other stimuli in vitro.
Pre-screens based on gene structure The relative ease in cloning the trapped gene allows the DNA sequence to be the criteria for prescreening genes of interest. For example, putative regulators of cell-cell interactions, transcription factors, molecules involved in signal transduction, etc., may be identified by their structural similarity to k n o w n genes. Gene-trap vectors that generate protein fusions are preferable for this purpose because the coding region of the endogenous gene may be found in RACE-generated cDNA clones. Cloning and sequencing of genes trapped in ES cells also has wider implications for genome mapping efforts. The localization of [3-galactosidase activity to a variety of subcellular compartments has been documented in gene-trap cell lines [5,9,10,25"] and may provide clues regarding the structure of the trapped gene. Normally, [3-galactosidase is distributed throughout the cytoplasm. The localization of [3-galactosidase to other subcellular structures presumably reflects the acquisition of endogenous protein domains that act as sorting signals. For example, a gene-trap fusion to a zinc finger containing protein shows [3-galactosidase activity in the nucleus of cells [12"]. The potential of the gene-trap approach to detect insertions in genes encoding secreted or transmembrane proteins has also been explored (WC Skarnes, RSP Beddington, unpublished data). Expression of a [3-galactosidase fusion containing a signal sequence has been found to lack enzymatic activity. Therefore, previous gene-trap screens have missed insertions into this class of genes. By including a transmembrane domain upstream of [3galactosidase in a gene-trap vector, enzymatic activity is restored in fusions with genes that encode aminoterminal signal sequences. Thus, insertions within cellsurface molecules are preferentially detected. Potential modifications in gene-trap vector designs may be envisioned to capture other classes of genes.
Pre-screens based on patterns of reporter gene expression Monitoring reporter gene activity in chimaeric embryos may be useful to find candidate genes relevant to a particular embryonic stage, developmental process, or cell lineage. Recently, 279 independent gene-trap insertions have been screened for l a c Z expression in chimaeric 8.5 day old embryos [25"]; 13 % of the cell lines showed restricted sites of expression, 32 % showed expression in most or all cells, and the remainder showed no expression. At least a third of the lines that showed no expression at 8.5 days were found to express l a c Z at later stages. Therefore, roughly two thirds of the genes expressed in ES cells display developmentally regulated patterns of expression in embryos. Simplified techniques for generating highly chimaeric embryos by morulae aggregation have b e e n developed [26"], which should increase the number of ES cell lines that can be analyzed in this way; however, a pre-screen based on expression patterns remains a significant undertaking. The differentiation of ES cell lines in vitro may provide
688
Mammalian gene studies an alternative method for identifying genes expressed in particular cell lineages. When grown in suspension, blastocyst-derived ES cells spontaneously give rise to a number of identifiable cell types including cardiac muscle, red blood cells, neurons, yolk-sac endoderm, and vascular endothelial cells [27,28]. Furthermore, defined culture conditions using various combinations of growth factors, have been established for the efficient and reproducible recovery of specific haematopoetic cell types [29]. A correlation of reporter gene expression with the appearance of specific cell types may help to pinpoint genes important in the differentiation of desired cell lineages.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest o, of outstanding interest 1.
CAPECCHIMR: Altering the G e n o m e by Homologous Recombination. Science 1989, 244:1288-1292.
2.
ROSSANTJ: Gene Disruption in Mammals. Curt Opin Genet Dev 1991, 1:236--240.
3.
JOYNERAL: Gone Targeting and Gene Trap Screens using Embryonic Stem Cells: New Approaches to Mammalian Development. Bioessays 1991, 13:649-656.
4.
SKARNESWC: Entrapment Vectors: A N e w Tool for Mammalian Genetics. Biotechnology 1990, 8:827-831.
5.
GOSSLERA, JOYNER AL, ROSSANTJ, SKARNESw e : Mouse Embryonic Stem Cells and Reporter Constructs to Detect Developmentally Regulated Genes. Science 1989, 244:463--465.
6.
VON MELCHNER H, REDDY S, RULEY HE: Isolation of Cellular Promoters by Using a Retrovirus Promoter Trap. Proc Natl Acad Sci USA 1990, 87:3733-3737.
7.
MACLEODD, LOVELL--BADGER, JONES S, .JACKSON I: A Prom o t e r Trap in Embryonic Stem (ES) Cells Selects for Integration of DNA into CpG Islands. Nucleic Acids Res 1991, 19:17-23.
8.
REDDYS, DEGREGOR!JV, VON MELCHNERH, RULEYHE: Retrovirus Promoter-Trap Vector to Induce lacZ Gene Fusions in Mammalian Cells. J Virol 1991, 65:1507-1515.
9.
BRENNERDG, L1N-CHAO S, COHEN SN: Analysis of Mammalian Cell Genetic Regulation in Situ by Using Retrovirus-Derived "Portable Exons ~ Carrying the Escherichia coil lacZ Gene. Proc Natl Acad Set USA 1989, 86:5517-5521.
10.
KERR WG, NOLAN GP, SERAFIN1 AT, HERZENBERG LA: Transcriptionally Defective Retroviruses Containing lacZ for t h e in Situ Detection of E n d o g e n o u s Genes and Developmentally Regulated Chromatin. Cold Spring Harb Syrup Quant Biol 1989, 54:767-776.
Conclusions
11.
In the past two years, the accumulated molecular and genetic data has validated the feasibility of gene trapping in ES cells to identify and mutate genes in the mouse. Thousands of gene-trap mutations can be conveniently obtained in culture a n d - - g i v e n the current rate of gemfline transmission and a reasonable amount of s p a c e - - i n the order of a hundred mutant mouse strains could be generated and analyzed by one laboratory over a period of two to three years. The rate of producing mutant mice by gene trapping is one to two orders of magnitude greater than that by targeted mutagenesis. Moreover, the development of prescreening strategies in ES cells should provide a versatile way to tailor screens towards specific interests.
FRIEDRICHG, SORIANOP: Promoter Traps in Embryonic Stem Cells: A Genetic Screen to Identify and Mutate Developmental Genes in Mice. Genes Dev 1991, 5:1513-1523.
SKARNESWC, AUERBACHA, JOYNER AL: A Gene Trap Approach in Mouse Embryonic Stem Cells: The lacZ Reporter is Activated by Splicing, Reflects Endogenous Gene Expression, and is Mutagenic in Mice. Genes Dev 1992, 6:903-918. Confirms all of the predictions o f the gene-trapping strategy. Using 5' RACE, the e n d o g e n o u s g e n e associated with three lacZ-based genetrap insertions is c k m e d and the proper use of the splice acceptor site is demonstrated. For two insertions, the pattern of lacZ expression in embryos is ~shown to match the normal distribution of e n d o g e n o u s transcripts. T w o of the three insertions tested cause phenotypic abnormalities in mice. O n e of these is an insertion into a novel g e n e expressed widely during development that causes peri-natal death in h o m o z y g o u s animals. The other is an insertion into a zinc-finger gene expressed in neural cells that results in mild growth retardation after birth.
Recently, one group has carried out a prescreen of gene-trap cell lines that are activated or repressed u p o n treatment with retinoic acid (L Forrester, W Wurst, personal communication). The effects of retinoic acid on axial patterning and limb development are well known, and genes responsive to retinoic acid nmy play a role in these processes. In a screen of 200 genetrap cell lines that expressed lacZ after treatment with retinoic acid for two days, expression was induced in nine cell lines and repressed in 11 cell lines. Many of these lines (70 %) showed spatially restricted expression in regions of the embryo known to be influenced by retinoic acid, that is, in the developing nervous system, limb buds and heart. Similar pre-screens, using a variety of other factors or morphogens, represent an exciting application of the gene-trap approach.
Acknowledgements I wish to thank L Forrester and W Wurst for communicating unpublished data, a n d L Forrester a n d A Smith for helpful c o m m e n t s and discussions. WC Skarnes is supported by an HFSP long-term fellowship.
12. e.
13. o•
VON MELCHNER H, DEGREGORI JV, RAYBURN H, REDDY S, FRIEDEL C, RULEY HE: Selective Disruption of Genes Expressed in Totipotent Embryonal Stem Cells. Genes Dev 1992, 6:919-927. Sequences upstream of nine retroviral promoter-trap insertions are cloned using inverse PCR. Flanking probes from five ES cell lines detected transcripts, and one clone is identified as the REX-1 transcription factor. T w o of four lines transmitted to the germline cause embryonic-lethM phenotypes. 14.
SHERIDEN U, RHODES K, BREINDI, M: Transcriptionally Active G e n o m e Regions are Preferred Targets for Retroviral Integration. Mol Cell Biol 1990, 64:907-912.
Gene trapping in transgenic m i c e Skarnes 15.
VIJAYAS, STEFFEN DL, ROBINSON HL: Acceptor Sites for Retro-
16.
ROHDEWOLDH, WEIHER H, REIK W, JAENISCH R, BREINDL H: Retrovirus Integration and Chromatin Structure: Moloney Murine Leukemia Proviral Integration Sites Map Near DNAse I Hypersensitive Sites. J Virol 1987, 61:336-343.
24.
viral Integrations Map Near DNase I-Hypersensitive Sites in Chromatin. J F~rol 1986, 60:683--692.
17.
BOGGS SS, GREGG RG, BORENSTEIN N, SMITHIES O: Efficient Transformation and Frequent Single Site, Single Copy Insertion of DNA can be Obtained in Mouse Erytholeukemia Cells Transformed by Electroporation. Exp Hematol 1986, 149:988--994.
18.
SOININENR, SCHOOR M, HENSELING U, TEPE C, KISTERS--WOIKE B, ROSSANTJ, GOSSLERA: The Mouse Enhancer Trap Locus 1 (Etl-1): A Novel Mammalian Gene Related to Drosophila and Yeast Transcriptional Regulator Genes. Mech Dev 1993, 39:111-123.
19.
NIWA H, ARAKI K, KIMURA S, TAN1GUCHI S, WAKASUG1 S, YAMAMUI~AK: An Efficient Gene-Trap Method using Poly A Trap Vectors and Characterization of Gene-Trap Events. J Btochem 1993, 113:343-349.
20.
BREINDL MK, HARBERS K, JAENISCH R: Retrovirus-Induced Lethal Mutation in Collagen I Gene of Mice is Associated with Altered Chromatin Structure. Cell 1984, 38:9-16.
21.
JAHNERD, JAENISCH R: Retrovirus-Induced de Novo Methylation of Flanking Host Sequences Correlates with Gene Inactivity. Nature 1985, 315:594-597.
22.
KRATOCHWILK, VON DER MARK K, KOLLAR E, JAENISCH R, MOOSLEHNER K, SCHWARTZM, MASSE K, GMACHL I, HARBERS K: Retroviral-Induced Mutation in Movl3 Mice Affects Collagen I Expression in a Tissue-Specific Manner. Cell 1989, 57:807-816.
23.
GOEBLMG, PETES TD: Most of the Yeast Genomic Sequences are Not Essential for Cell Growth and Division. Cell 1986, 46:983-922.
WILSONC, KURTH R, BELLEN MJ, O'KANE CJ, GROSSNIKLAUS U, GEHRING WJ: P-Element-Mediated Enhancer Detection: An Efficient Method for Isolating and Characterizing Developmentally Regulated Genes in Drosophila. Genes Dev 1989, 3:1301-1313.
25. •
WURSTW, ROSSANTJ, PRIDEAUX V, KOWNACKAM, JOYNER A, HILL D, GUILLEMOT F, GASCA S, AUERBACH A, ANG S-L: An Embryonic Expression Screen for Spatially Restricted Genes using Gcne Trap Integrations in ES Cell Chimaeras. Mech Dev 1993, in press. A screen o f 279 gene-trap cell lines for expression in 8.5 day embryos reveals developmentally regulated patterns in two thirds of the cases. 26. -
WOOD SA, ALLENND, ROSSANTJ, AUERBACHA, NAGY A: NonInjection Methods for the Production of Embryonic Stem Cell-Embryo Chimaeras. Nature 1993, 365:87-89. Describes time-saving m e t h o d s for generating ES cell derived chimaeras by morulae agg~regation. These can be used to more easily carry out a prescreen based on embryonic expression of the reporter.
27.
DOETSCHMANTC, EISTE+ITER H, KATZ M, SCHMIDT W, KELLER R: The in Vitro Development of Blastocyst-Derived Embryonic Stem Cell Lines: Formation of Visceral Yolk Sac, Blood Islands and Myocardium. J Emb~ol Exp Morph 1985, 87:27~i5.
28.
RISAUW, SARIOLAH, ZERWES MG, SASSEJ, EKBLOM P, KEMLER R, DOETSCHMAN T: Vasculogenesis and Anglogenesis in Embryonic-Stem-Cell-Derived Embryoid Bodies. Development 1988, 102:471-478.
29.
WILES MV, KELLER G: Multiple Hematopoetic Lineages Develop from Embryonic Stem (ES) Cells in Culture. Development 1991, 111:259-267.
WC Skarnes, AFRC Centre for G e n o m e Research, King's Buildings, West Mains Road, Edinburgh EH9 3JQ, UK.
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