In vitro preselection of gene-trapped embryonic stem cell clones for characterizing novel developmentally regulated genes in the mouse
In vitro preselection of gene-trapped embryonic stem cell clones for characterizing novel developmentally regulated genes in the mouse
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DOI：
10.1006/dbio.1997.8541
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发表时间：
1997-05-15
影响因子：
2.7
通讯作者：
Lyons, GE
中科院分区：
文献类型：
--
作者：
Baker, RK;Haendel, MA;Lyons, GE
我们已经开发出一种体外基因陷阱筛选新的小鼠基因，允许人们在制造嵌合或转基因动物之前确定这些基因是否在一个或多个特定的胚胎组织中表达。全能胚胎干细胞被编码可选择的IacZ/neo(R)融合基因的逆转录病毒基因陷阱构建体感染，只有当基因陷阱插入到激活的转录单位中时才能表达。G418抗性ES细胞克隆在体外被诱导分化，并对神经元、神经胶质细胞、心肌细胞和软骨细胞进行β-半乳糖苷酶(β-GAL)的表达筛选。通过5‘端快速扩增获得基因陷阱转录本的cDNAs，并对其进行测序，以确定它们是否代表新基因。原位杂交分析表明，捕获的基因在体内表达在体外表达β-半乳糖的细胞类型中。基因陷阱及其野生型等位基因的特征是拷贝数、转录本的交替剪接以及杂交基因陷阱转录本中被IacZ/neo(R)替换的内源mRNA序列的比例。这种方法，我们称之为“体外预选”，比标准的体内基因陷阱筛选更经济，因为在产生转基因动物之前，可能的敲除等位基因的组织特异性表达是被验证的。这些结果也强调了胚胎干细胞体外分化作为一种研究调节各种细胞和组织类型的规格和承诺的分子机制的方法的实用性。(C)1997年学术出版社。
We have developed an in vitro gene trap screen for novel murine genes that allows one to determine, prior to making chimeric or transgenic animals, if these genes are expressed in one or more specific embryonic tissues. Totipotent embryonic stem (ES) cells are infected with a retroviral gene trap construct encoding a selectable IacZ/neo(R) fusion gene, which is expressed only if the gene trap inserts within an active transcription unit. G418-resistant ES cell clones are induced to differentiate in vitro, and neurons, glia, myocytes, and chondrocytes are screened for expression of beta-galactosidase (beta-gal). cDNAs of the gene trap transcripts are obtained by 5' rapid amplification of cDNA ends and are sequenced to determine if they represent novel genes. In situ hybridization analyses show that trapped genes are expressed in vivo within the cell types that express beta-gal in vitro. Gene traps and their wild-type alleles are characterized in terms of copy number, alternate splicing of their transcripts, and the proportion of endogenous mRNA sequence that is replaced by IacZ/neo(R) in the hybrid gene trap transcript. This approach, which we term ''in vitro preselection,'' is more economical than standard in vivo gene trap screening because tissue-specific expression of probable knockout alleles is verified before transgenic animals are generated. These results also highlight the utility of ES cell differentiation in vitro as a method with which to study the molecular mechanisms regulating the specification and commitment of a variety of cell and tissue types. (C) 1997 Academic Press.