Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells.

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.     

EUCOMM--the European conditional mouse mutagenesis program.

Functional analysis of the mammalian genome is an enormous challenge for biomedical scientists. To facilitate this endeavour, the European Conditional Mouse Mutagenesis Program (EUCOMM) aims at generating up to 12 000 mutations by gene trapping and up to 8000 mutations by gene targeting in mouse embryonic stem (ES) cells. These mutations can be rendered into conditional alleles, allowing Cre recombinase-mediated disruption of gene function in a time- and tissue-specific manner. Furthermore, the EUCOMM program will generate up to 320 mouse lines from the EUCOMM resource and up to 20 new Cre driver mouse lines. The EUCOMM resource of vectors, mutant ES cell lines and mutant mice will be openly available to the scientific community. EUCOMM will be one of the cornerstones of an international effort to create a global mouse mutant resource.     

Requirement of N-myc protein down-regulation for neuronal differentiation in the spinal cord

Previous work has indicated that N-myc expression occurs widely in the developing central nervous system, but its level changes dynamically with region- and stage-specificities. We show in the present report that in the developing spinal cord of the mouse, N-myc protein expression takes place in the ventricular zone and reaches its maximum at the outermost layer, but is extinct in the intermediate zone, indicating that N-myc protein is not expressed in mature neurons. We examined the effect of forced, persistent N-myc expression in development of the spinal cord in order to understand the functional significance of N-myc down-regulation. We made embryonic stem (ES) cell lines that constitutively expressed N-myc at a high level, then produced mouse embryo chimeras with a high contribution of the ES cells. The majority of the chimeras developed to day 12 with normal gross morphology, but in these chimeras neuronal differentiation in the spinal cord was perturbed at the histological level. Intermediate zones and ventral horns were formed, but the expression of N-CAM and neurofilaments was diminished. Chimeras using β-galactosidase-expressing recipient embryos indicated that inhibition of the neuronal differentiation was a cell-autonomous effect of persistent N-myc expression. These observations indicate that N-myc down-regulation in individual cells is required for full differentiation of neurons.     

The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes.

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.     

低钾型周期性麻痹相关的Cchl1a3基因R528H敲入小鼠模型的构建

目的：构建低钾型周期性麻痹相关的Cchl1a3基因R528H敲入小鼠模型。方法将 Cchl1a3-knock-in打靶载体电转染ES细胞,经过G418和Ganciclovoir筛选阳性ES细胞克隆并用PCR和DNA测序法鉴定。将阳性ES克隆注射到小鼠囊胚,获得嵌合体小鼠。通过杂交获得的杂合子小鼠与FLP小鼠交配繁育获得去neo杂合子小鼠,并用PCR和DNA测序进行鉴定。将去neo杂合子小鼠交配得到纯合子后代,进行生长发育等方面的观察。结果打靶载体成功转染ES细胞,PCR和DNA测序法证实9个ES细胞克隆发生正确的同源重组。通过显微注射获得7只嵌合体小鼠。将嵌合体小鼠交配繁育的杂合子小鼠和FLP小鼠交配获得9只去neo杂合子小鼠,最终得到15只去neo纯合子小鼠。该小鼠在发育至性成熟阶段,精神、饮食及活动状态良好,但是在4个月龄时逐渐出现脱毛,皮肤破溃甚至死亡。结论成功构建Cchl1a3基因 R528H 突变的纯合子小鼠,为研究人类CACNA1S基因功能和阐明低钾型周期性麻痹发生的分子机制奠定了基础。     

Retention of the Developmental Pluripotency in Medaka Embryonic Stem Cells after Gene Transfer and Long-term Drug Selection for Gene Targeting in Fish

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.     

Generation of an Fgf9 conditional null allele

The fibroblast growth factor (FGF) family consists of 22 widely expressed regulatory polypeptides and controls a broad spectrum of cellular processes. Accumulating data show that FGF9 plays important roles both in embryogenesis and in adult tissue homeostasis. Ablation of Fgf9 alleles leads to lethality at the neonatal stage mainly due to malformations of the lung, as well as causing male-to-female sex reversal. To circumvent the neonatal lethality resulting from disruption of the Fgf9 gene, which hinders further characterization of the role of FGF9 in adult tissue function and homeostasis, we generated an Fgf9 conditional null allele for spatiotemporal- and tissue-specific disruption of Fgf9. Using gene targeting in mouse embryonic stem (ES) cells, we introduced two loxP sites flanking exon 1 in the Fgf9 allele, which encodes 93 amino acid residues at the N-terminal of FGF9. Our results indicate that the Fgf9 conditional null allele is a true conditional null that encodes wildtype activity and reverts to a null allele after recombination mediated by the Cre recombinase.     

Production of homozygous mutant ES cells with a single targeting construct.

We have developed a simple method for producing embryonic stem (ES) cell lines whereby both alleles have been inactivated by homologous recombination and which requires a single targeting construct. Four different ES cell lines were created that were heterozygous for genes encoding two guanine nucleotide-binding protein subunits, alpha i2 and alpha i3, T-cell receptor alpha, and beta-cardiac myosin heavy chain. When these heterozygous cells were grown in high concentrations of G418, many of the surviving cells were homozygous for the targeted allele and contained two copies of the G418 resistance gene. This scheme provides an easy method for obtaining homozygous mutationally altered cells, i.e., double knockouts, and should be generally applicable to other genes and to cell lines other than ES cells. This method should also enable the production of cell lines in which more than one gene have had both alleles disrupted. These mutant cells should provide useful tools for defining the role of particular genes in cell culture.     

BPOZ基因剔除小鼠模型的建立

BPOZ是在卵巢癌等肿瘤组织中表达下调的细胞生长抑制基因,建立BPOZ基因剔除小鼠模型,可以为在体研究BPOZ基因的生物学功能及其与肿瘤发生的关系创造条件.运用生物信息学手段确定小鼠BPOZ基因组序列,设计基因剔除策略,构建完成了基因剔除载体XpPNT-BPOZ.以电穿孔方法将基因剔除载体导入ES细胞,用G418和Ganciclovoir进行正负筛选,获得抵抗克隆,PCR和DNA印迹鉴定出正确同源重组的ES细胞克隆.将同源重组的ES细胞注入小鼠囊胚,获得嵌合体小鼠.嵌合体小鼠与C57BL/6J小鼠交配后获得Aguoti毛色的小鼠30只,其中15只为BPOZ基因剔除杂合子小鼠,阳性率为50%.在雌雄杂合子交配的后代中获得纯合子小鼠.初步的表型观察发现BPOZ基因剔除小鼠发育正常,有繁殖能力,进一步的表型分析工作正在进行之中.     

Forced expression of the homeobox-containing gene Pem blocks differentiation of embryonic stem cells.

Similarities in the differentiation of mouse embryos and ES cell embryoid bodies suggest that aspects of early mammalian embryogenesis can be studied in ES cell embryoid bodies. In an effort to understand the regulation of cellular differentiation during early mouse embryogenesis, we altered the expression of the Pem homeobox-containing gene in ES cells. Pem is normally expressed in the preimplantation embryo and expressed in a lineage-restricted fashion following implantation, suggesting a role for Pem in regulating cellular differentiation in the early embryo. Here, we show that the forced expression of Pem from the mouse Pgk-1 promoter in ES cells blocks the in vitro and in vivo differentiation of the cells. In particular, embryoid bodies produced from these Pgk-Pem ES cells do not differentiate into primitive endoderm or embryonic ectoderm, which are prominent features of early embryoid bodies from normal ES cells. This Pgk-Pem phenotype is also different from the null phenotype, as embryoid bodies derived from ES cells in which endogenous Pem gene expression has been blocked show a pattern of differentiation similar to that of normal ES cells. When the Pgk-Pem ES cells were introduced into subcutaneous sites of nude mice, only undifferentiated EC-like cells were found in the teratomas derived from the injected cells. The Pem-dependent block of ES cell differentiation appears to be cell autonomous; Pgk-Pem ES cells did not differentiate when mixed with normal, differentiating ES cells. A block to ES cell differentiation, resulting from the forced expression of Pem, can also be produced by the forced expression of the nonhomeodomain region of Pem. These studies are consistent with a role for Pem in regulating the transition between undifferentiated and differentiated cells of the early mouse embryo.     

Lack of beta 1 integrin gene in embryonic stem cells affects morphology, adhesion, and migration but not integration into the inner cell mass of blastocysts

A gene trap-type targeting vector was designed to inactivate the beta 1 integrin gene in embryonic stem (ES) cells. Using this vector more than 50% of the ES cell clones acquired a disruption in the beta 1 integrin gene and a single clone was mutated in both alleles. The homozygous mutant did not produce beta 1 integrin mRNA or protein, while alpha 3, alpha 5, and alpha 6 integrin subunits were transcribed but not detectable on the cell surface. Heterozygous mutants showed reduced beta 1 expression and surface localization of alpha/beta 1 heterodimers. The alpha V subunit expression was not impaired on any of the mutants. Homozygous ES cell mutants lacked adhesiveness for laminin and fibronectin but not for vitronectin and showed a reduced association with a fibroblast feeder layer. Furthermore, they did not migrate towards chemoattractants in fibroblast medium. None of these functions were impaired in heterozygous mutants. Scanning electron microscopy revealed that homozygous cells showed fewer cell-cell junctions and had many microvilli not usually found on wild type and heterozygous cells. This profound change in cell shape is not associated with gross alterations in the expression and distribution of cytoskeletal components. Unexpectedly, microinjection into blastocysts demonstrated full integration of homozygous and heterozygous mutants into the inner cell mass. This will allow studies of the consequences of beta 1 integrin deficiency in several in vivo situations.     

Cre-mediated recombination at the murine whey acidic protein (mWAP) locus

The mouse whey acidic protein (WAP) gene in mouse embryonic stem (ES) cells has been targeted with a loxP-flanked neomycin phosphotransferase-thymidine kinase (neo-TK) cassette inserted into exon 4. Southern blot revealed that 51 of 199 colonies were correctly targeted (1:4). Next, a Cre-encoding plasmid was electroporated into a targeted cell line to cause the deletion of the neo-TK cassette. Modified ES cell colonies were identified by polymerase chain reaction (PCR); 44 out of 50 colonies (88%) had undergone Cre-mediated deletion. Finally, a loxP-tagged cell line was co-electroporated with a Cre-encoding plasmid and a loxP-containing neo plasmid for site-specific insertion into the WAP locus. The frequency of this event was 23% (11 of 48) of that obtained with random integration. This demonstrates the feasibility of using the Cre-loxP system for site-specific integration in ES cells. Moreover, this is the first report of targeting a loxP-containing transgene into a predetermined location in ES cells. Ultimately, a mouse model derived from these modified ES cells will usher in a second generation of animal “bioreactor” models where the inserted transgene is controlled exclusively by the endogenous locus regulatory elements. In addition, oncogenesis can be explored from single copy oncogene/tumor suppressor gene inserts, which are regulated in a temporal and tissue-specific manner. It is hoped that regulation of transgene expression in this fashion will help elucidate the underlying mechanisms of normal development in the mammary gland. Mol. Reprod. Dev. 48:324–331, 1997. © 1997 Wiley-Liss, Inc.     

p35Nck5a基因重复性打靶载体的构建和ES细胞基因打靶研究

为了在小鼠胚胎于细胞（ＥＳ）中引起神经细胞ｃｄｃ２类激酶调节亚基ｐ３５Ｎｃｋ５ａ基因的定点 重复,采用常规的分子克隆技术,构建得到长约１２．２ｋｂ的基因重复性打靶载体ｐＧＤＴＶ。用电 穿孔法将线性化的ｐＧＤＴＶ载体转入ＥＳ细胞,经过Ｇ４１８和ＧＡＮＣ分组药物选择,获得２４５个 双药物抗性的细胞克隆,细胞存活率为６．２２ × １０－５。经ＰＣＲ和基因组Ｓｏｕｔｈｅｒｎ杂交鉴定,２个 ＥＳ细胞克隆发生了ｐ３５Ｎｃｋ５ａ基因的重复,同源重组率为５．０８×１０－７、负向选择系统的应用使 同源重组事件的富集效率提高了７倍。为建立Ａｌｚｈｅｉｍｅｒ病的转基因小鼠模型打下了基础。     

Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells

We mutated, by gene targeting, the endogenous hypoxanthine phosphoribosyl transferase (HPRT) gene in mouse embryo-derived stem (ES) cells. A specialized construct of the neomycin resistance (neor) gene was introduced into an exon of a cloned fragment of the Hprt gene and used to transfect ES cells. Among the G418r colonies, 1/1000 were also resistant to the base analog 6-thioguanine (6-TG). The G418r, 6-TGr cells were all shown to be Hprt- as the result of homologous recombination with the exogenous, neor-containing, Hprt sequences. We have compared the gene-targeting efficiencies of two classes of neor-Hprt recombinant vectors: those that replace the endogenous sequence with the exogenous sequence and those that insert the exogenous sequence into the endogenous sequence. The targeting efficiencies of both classes of vectors are strongly dependent upon the extent of homology between exogenous and endogenous sequences. The protocol described herein should be useful for targeting mutations into any gene.     

Rapid construction in yeast of complex targeting vectors for gene manipulation in the mouse.

Targeting vectors for embryonic stem (ES) cells typically contain a mouse gene segment of >7 kb with the neo gene inserted for positive selection of the targeting event. More complex targeting vectors carry additional genetic elements (e.g. lacZ, loxP, point mutations). Here we use homologous recombination in yeast to construct targeting vectors for the incorporation of genetic elements (GEs) into mouse genes. The precise insertion of GEs into any position of a mouse gene segment cloned in an Escherichia coli/yeast shuttle vector is directed by short recombinogenic arms (RAs) flanking the GEs. In this way, complex targeting vectors can be engineered with considerable ease and speed, obviating extensive gene mapping in search for suitable restriction sites.