Embryonic stem cells stably transfected with mRAR beta 2-lacZ exhibit specific expression in chimeric embryos.

Using the embryonic stem (ES) cell/chimera approach, we have studied the activity of the mouse retinoic acid receptor beta 2 (mRAR beta 2) promoter during ES cell differentiation and during embryonic development. Stable ES clones were isolated after introduction of a 1.8 kb mRAR beta 2-lacZ expression cassette. LacZ expression in these stable clones was specifically induced by retinoic acid (RA) in a similar fashion as the endogenous RAR beta 2 gene. Following introduction of three different ES clones into blastocysts, an integration-independent mRAR beta 2-lacZ expression pattern was obtained in chimeric embryos similar to that described by in situ hybridization and transgenic studies. Moreover, mRAR beta 2-lacZ expression was also detected at some additional sites not described before, e.g. body wall, ureter, mesonephric duct and optic stalk. Maternal RA administration at 8.5 days of pregnancy extended lacZ expression to more anterior and posterior regions. Transgenic mice were generated from germ-line transmission of the transfected ES cells; expression pattern and changes in expression upon RA induction in these transgenic embryos were identical to those in chimeric embryos. We conclude that by using the ES/chimera approach, the proximal 1.8 kb of the mRAR beta 2 promoter produces a reliable and reproducible expression pattern of the reporter gene, and that the ES cell/chimera approach is invaluable for the study of gene expression and regulation.     

Unexpected behavior of a gene trap vector comprising a fusion between the Sh ble and the lacZ genes

A new gene trap vector has been designed, comprised of a fusion between the Sh ble gene, which confers resistance to the antibiotic phleomycin, and the lacZ gene (phleal fusion gene). A synthetic splice acceptor, made of the yeast branchpoint followed by a pyrimidin-rich sequence of 27 nucleotides, is included at the 5′ extremity. The linearized gene trap vector was introduced into mouse embryonic stem cells (ES cells), and 40 phleomycin resistant (phleAL) cell lines possessing a single copy of the insert were selected. They were stable in expressing the lacZ gene. Reporter gene expression was studied at days 8.5 and 10.5 of embryonic development in chimeric embryos obtained after injection of phleoL ES clones into 8-cell stage embryos. Out of 20 phleal lines examined, 14 exhibited β-galactosidase expression at day 10.5. Use of the phleal fusion gene trap vector to select genes expressed in ES cells, therefore, is compatible with the isolation of genes expressed at midgestation. However, and most intriguingly, 10 out of these 14 cell lines (71%) displayed reporter gene expression mostly in heart and liver. Two of them exhibited, in addition, expression in central nervous system (CNS) or in CNS and limb buds, respectively. Germline chimeras were subsequently obtained and 15 mouse lines have been established. Intercrosses of animals heterozygous for the insertion revealed a mutant phenotype in several lines. © 1996 Wiley-Liss, Inc.     

Sequence and expression of a novel mouse gene PRDC (protein related to DAN and cerberus) identified by a gene trap approach

Gene trapping in embryonic stem (ES) cells was used to identify a novel gene involved in mouse development. In order to screen trapped ES cell lines for the presence of developmentally regulated genes, an in vitro differentiation test was used. One of the G418 resistant cell lines, in conjunction with the lacZ reporter gene, showed differential expression patterns under differentiated and undifferentiated conditions. The gene trap insertion in this cell line was germ-line transmitted and X-gal staining was used to assess the expression pattern of lacZ in embryos heterozygous for the trapped allele. The reporter gene's expression was detected in commissural neurons in the developing spinal cord, suggesting functions for the trapped gene in mouse neural development. Structural analysis of the cDNA revealed that this trapped gene, named PRDC (protein related to DAN and cerberus), is a novel gene that encodes a putative secretory protein consisting of 168 amino acid residues. PRDC gene product shows limited similarities to the products of DAN (differential screening-selected gene aberrative in neuroblastoma) and cerberus . (DAN is a possible tumor-suppressor for neuroblastoma in human. Cerberus can induce an ectopic head in Xenopus embryos when ectopically expressed.) These three gene products may form a novel family of signaling molecules.     

An alternative simple method for mass production of chimeric embryos by coculturing denuded embryos and embryonic stem cells in Eppendorf vials

The generation of germline competent chimeric mice via embryonic stem (ES) cells is a crucial step in developing gene-manipulated mouse models. To date, techniques for generating chimeric mice include direct microinjection of ES cells into the cavity of 3.5-d post-coitum (dpc) blastocysts and aggregating or coculturing 2.5 dpc zona pellucida-free (denuded) embryos with ES cells. We present here a procedure that is simple and reproducible for mass producing (10-150 embryos/vial/time) chimeric embryos by coculturing denuded 8-cell embryos and morula in 0.8 mL KSOM-AA medium containing 5 x 10(5)mL-1 purified green fluorescence protein-expressing ES cells (either fresh or thawed) in an 1.7 mL Eppendorf vial for 3h. The resulting chimeras had substantial levels of chimerism and high germline transmission rates. Therefore, the method developed in this study can provide a simple and mass reproducible alternative method (to germline transmitter chimeric mice), without technological and instrumental difficulties, for generating chimeric embryos.     

DNA methylation-mediated control of Sry gene expression in mouse gonadal development

DNA methylation at CpG sequences is involved in tissue-specific and developmentally regulated gene expression. The Sry (sex-determining region on the Y chromosome) gene encodes a master protein for initiating testis differentiation in mammals, and its expression is restricted to gonadal somatic cells at 10.5-12.5 days post-coitum (dpc) in the mouse. We found that in vitro methylation of the 5'-flanking region of the Sry gene caused suppression of reporter activity, implying that Sry gene expression could be regulated by DNA methylation-mediated gene silencing. Bisulfite restriction mapping and sodium bisulfite sequencing revealed that the 5'-flanking region of the Sry gene was hypermethylated in the 8.5-dpc embryos in which the Sry gene was not expressed. Importantly, this region was specifically hypomethylated in the gonad at 11.5 dpc, while the hypermethylated status was maintained in tissues that do not express the Sry gene. We concluded that expression of the Sry gene is under the control of an epigenetic mechanism mediated by DNA methylation.     

Generation of multipurpose alleles for the functional analysis of the mouse genome.

A novel generation of retroviral gene-trap vectors has been developed with the ability to induce conditional mutations in most genes expressed in mouse embryonic stem (ES) cells. The vectors rely on directional site-specific recombination systems, which can repair and re-induce the gene-trap mutations when activated in succession. After the gene-trap insertions are passaged into mice, this system enables the induction of temporally and spatially restricted mutations in somatic cells. In addition to their conditional features, the vectors create multipurpose alleles amenable to a wide range of post-insertional modifications. These vectors have been used to assemble the largest library of ES cell lines with conditional mutations in single genes, presently totalling 1724 unique genes. Due to their efficiency, the vectors are part of the core technologies to be used by EUCOMM for establishing a complete collection of conditional null mutations in mice.     

介绍一种鉴定胚胎干细胞基因打靶快速、经济的筛选新方法

小鼠基因剔除动物模型越来越广泛地应用于哺乳动物基因功能与疾病的研究。然而每当胚胎干细胞同源重组的效率过低时,鉴定与分离带有定位变异的阳性克隆就会既费力又昂贵。本工作以类固醇受体共激活子基因为例,研究出一种快速鉴定阳性克隆的新方法。在构造重组载体时,将一段编码半乳糖苷酶的DNA序列整合到共激活子基因的蛋白起始码后面。于是,在干细胞内同源重组发生以后,半乳糖苷酶的表达就会受控于内源性共激活子基因的启动子。在载体与半乳糖苷酶DNA随机整合的大多数非特异克隆中,因为缺少启动子或由于不正确的氨基酸编码连接,导致合成半乳糖苷酶的可能性较小。因此,在半乳糖苷酶染色阳性的克隆中,具有特异突变的阳性克隆可以富集30倍以上。从半乳糖苷酶的阳性克隆中,再用Southern Blot方法进一步确认带有基因剔除的阳性克隆就大大减少了工作量。因为半乳糖苷酶的细胞化学染色法简便而可靠,所以在重组效率低时,可以用这种方法在短期内筛选大量克隆。但是应该注意,应用该方法的前提条件是所研究的基因必须在胚胎干细胞内表达。这些方法更为重要的意义在于当带有基因剔除的胚胎干细胞发育成小鼠后,半乳糖苷酶的组化染色法可以轻而易举地用来揭示所研究基因在动物不同组织与细胞中的表达水平。     

A high-throughput induction gene trap approach defines C4ST as a target of BMP signaling

Here we describe a novel gene trap protocol to screen for target genes that are regulated during inductive events in undifferentiated and differentiated mouse embryonic stem cells. This approach integrates several features that allows in vitro screening of large numbers of gene trap clones prior to generating lines of mutant mice. Moreover, targets of spatially and temporally restricted signaling pathways can be analyzed by screening undifferentiated ES cells versus ES cells differentiated into embryoid bodies. We employed this protocol to screen 1920 gene trap lines to identify targets and mediators of signaling through three growth factors of the TGFbeta superfamily--BMP2, activin and nodal. We identified two genes that are induced by BMP2 in a differentiation-dependent manner. One of the genes encodes for Chondroitin-4-sulfotransferase and displays a highly specific temporal and spatial expression pattern during mouse embryogenesis. These results demonstrate the feasibility of a high-throughput gene trap approach as a means to identify mediators and targets of multiple growth factor signaling pathways that function during different stages of development.     

Gene-trap mutagenesis: past, present and beyond

Although at least 35,000 human genes have been sequenced and mapped, adequate expression or functional information is available for only approximately 15% of them. Gene-trap mutagenesis is a technique that randomly generates loss-of-function mutations and reports the expression of many mouse genes. At present, several large-scale, gene-trap screens are being carried out with various new vectors, which aim to generate a public resource of mutagenized embryonic stem (ES) cells. This resource now includes more than 8,000 mutagenized ES-cell lines, which are freely available, making it an appropriate time to evaluate the recent advances in this area of genomic technology and the technical hurdles it has yet to overcome.     

Inducible gene trapping with drug-selectable markers and Cre/loxP to identify developmentally regulated genes

Gene trapping in mouse embryonic stem cells is an important genetic approach that allows simultaneous mutation of genes and generation of corresponding mutant mice. We designed a selection scheme with drug selection markers and Cre/loxP technology which allows screening of gene trap events that responded to a signaling molecule in a 96-well format. Nine hundred twenty gene trap clones were assayed, and 258 were classified as gene traps induced by in vitro differentiation. Sixty-five of the in vitro differentiation-inducible gene traps were also responsive to retinoic acid treatment. In vivo analysis revealed that 85% of the retinoic acid-inducible gene traps trapped developmentally regulated genes, consistent with the observation that genes induced by retinoic acid treatment are likely to be developmentally regulated. Our results demonstrate that the inducible gene trapping system described here can be used to enrich in vitro for traps in genes of interest. Furthermore, we demonstrate that the cre reporter is extremely sensitive and can be used to explore chromosomal regions that are not detectable with neo as a selection cassette.     

A Cre recombinase transgene with mosaic, widespread tamoxifen-inducible action

Cre-mediated site-specific recombination allows conditional transgene expression or gene knockouts in mice. Inducible Cre recombination systems have been developed to bypass initial embryonic lethal phenotypes and provide access to later embryonic or adult phenotypes. We have produced Cre transgenic mice in which excision is tamoxifen inducible and occurs in a widespread mosaic pattern. We utilized our Cre excision reporter system combined with an embryonic stem (ES) cell screen to identify ES cell clones with undetectable background Cre activity in the absence of tamoxifen but efficient excision upon addition of tamoxifen. The CreER transgenic mouse lines derived from the ES cells were tested using the Z/AP and Z/EG Cre reporter lines. Reporter gene expression indicated Cre excision was maximal in midgestation embryos by 2 days after tamoxifen administration, with an overall efficiency of 5-10% of cells with Cre excision. At 3 days after tamoxifen treatment most reporter gene expression marked groups of cells, suggesting an expansion of cells with Cre excision, and the proportion of cells with Cre excision was maintained. In adults, Cre excision was also observed with varying efficiencies in all tissues after tamoxifen treatment.