Gene trap mutagenesis of hnRNP A2/B1:: a cryptic 3′ splice site in the neomycin resistance gene allows continued expression of the disrupted cellular gene -: art. no. 2
Gene trap mutagenesis of hnRNP A2/B1:: a cryptic 3′ splice site in the neomycin resistance gene allows continued expression of the disrupted cellular gene -: art. no. 2
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DOI：
10.1186/1471-2164-4-2
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发表时间：
2003-01-20
期刊：
影响因子：
4.4
通讯作者：
Ruley, HE
中科院分区：
文献类型：
--
作者：
Roshon, M;DeGregori, JV;Ruley, HE
背景资料：标记序列诱变是一种在胚胎干细胞中构建测序插入突变文库的方法，这些突变可以传递到小鼠生殖系中。为了更好地预测基因截留对细胞基因表达的功能后果，本研究的特点是插入hnRNPA 2/B1基因内含子的U3 Neo基因陷阱逆转录病毒的影响。选择突变进行分析，因为它发生在一个高表达的基因，但没有产生明显的表型以下生殖系transmission.Results：侧翼的整合基因陷阱载体在IB 4细胞中的序列被用来分离全长cDNA，其预测的氨基酸序列是相同的人A2蛋白在所有，但341个氨基酸残基之一。hnRNP A2/B1转录物延伸到前病毒中，利用位于新霉素磷酸转移酶起始密码子下游28个核苷酸的隐蔽3'剪接位点。插入的Neo序列和前病毒poly（A）位点作为3'末端外显子起作用，其用于产生hnRNP A2/B1-Neo融合转录物，或跳过以产生野生型hnRNP A2/B1转录物。结论：hnRNPA 2/B1基因的表达和病毒poly（A）位点的利用符合前体mRNA剪接的外显子定义模型。这些结果揭示了一种机制，通过该机制，U3基因诱捕载体可以在不破坏细胞基因表达的情况下表达，从而提出了改进这些载体用于基因诱捕诱变的方法。
Background: Tagged sequence mutagenesis is a process for constructing libraries of sequenced insertion mutations in embryonic stem cells that can be transmitted into the mouse germline. To better predict the functional consequences of gene entrapment on cellular gene expression, the present study characterized the effects of a U3Neo gene trap retrovirus inserted into an intron of the hnRNP A2/B1 gene. The mutation was selected for analysis because it occurred in a highly expressed gene and yet did not produce obvious phenotypes following germline transmission.Results: Sequences flanking the integrated gene trap vector in IB4 cells were used to isolate a full-length cDNA whose predicted amino acid sequence is identical to the human A2 protein at all but one of 341 amino acid residues. hnRNP A2/B1 transcripts extending into the provirus utilize a cryptic 3' splice site located 28 nucleotides downstream of the neomycin phosphotransferase start codon. The inserted Neo sequence and proviral poly( A) site function as an 3' terminal exon that is utilized to produce hnRNP A2/B1-Neo fusion transcripts, or skipped to produce wild-type hnRNP A2/B1 transcripts. This results in only a modest disruption of hnRNPA2/B1 gene expression.Conclusions: Expression of the occupied hnRNP A2/B1 gene and utilization of the viral poly( A) site are consistent with an exon definition model of pre-mRNA splicing. These results reveal a mechanism by which U3 gene trap vectors can be expressed without disrupting cellular gene expression, thus suggesting ways to improve these vectors for gene trap mutagenesis.