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
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
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DOI：
10.1101/gad.6.6.903
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发表时间：
1992-06-01
影响因子：
10.5
通讯作者：
JOYNER, AL
中科院分区：
文献类型：
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作者：
SKARNES, WC;AUERBACH, BA;JOYNER, AL
我们已经证实，基因捕获载体pGT4.5会产生与内源基因剪接的融合转录本，并阻止整合位点正常转录本的合成。在三个 ES 细胞系中制备 lacZ 融合转录物的 cDNA，以恢复 lacZ 上游的内源外显子序列。每个克隆都检测到独特大小的内源转录物，以及该克隆所源自的 ES 细胞系中的融合转录物。对这些克隆和从随机引发的 cDNA 文库中分离的较大克隆进行的序列分析表明，剪接受体使用得当。对于两个插入，在三个胚胎阶段对 lacZ 报告基因和相关内源基因的表达模式进行了原位比较，发现相似。三个基因陷阱插入被传递到种系中，并且三个插入中的两个在纯合状态下观察到异常。对从两个突变基因陷阱插入的纯合小鼠获得的 RNA 进行了正常内源转录本的分析，检测到的量可以忽略不计，表明基因陷阱插入周围几乎没有发生剪接。这项工作证明了基因捕获载体生成 lacZ 融合转录本、准确报告内源基因表达以及在整合位点突变内源基因的能力。
We have confirmed that the gene trap vector pGT4.5 creates spliced fusion transcripts with endogenous genes and prevents the synthesis of normal transcripts at the site of integration. cDNA was prepared to the lacZ fusion transcript in three ES cell lines to recover endogenous exon sequences upstream of lacZ. Each of the clones detected a unique-sized endogenous transcript, as well as the fusion transcript in the ES cell line from which the clone was derived. Sequence analysis of these clones and larger clones isolated from a random-primed cDNA library showed that the splice acceptor was used properly. For two insertions, the expression patterns of the lacZ reporter and the associated endogenous gene were compared in situ at three embryonic stages and were found to be similar. Three gene trap insertions were transmitted into the germ line, and abnormalities were observed with two of the three insertions in the homozygous state. RNA obtained from mice homozygous for the two mutant gene trap insertions was analyzed for normal endogenous transcripts and negligible amounts were detected, indicating that little splicing around the gene trap insertion occurred. This work demonstrates the capacity of the gene trap vector to generate lacZ fusion transcripts, to accurately report endogenous gene expression, and to mutate the endogenous gene at the site of integration.