Yang Shao H, Bergo Martin O, Farber Emily, Qiao Xin, Fong Loren G, Young Stephen G
Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
Transgenic Res. 2009 Jun;18(3):483-9. doi: 10.1007/s11248-008-9237-9. Epub 2008 Dec 18.
A common strategy for conditional knockout alleles is to "flox" (flank with loxP sites) a 5' exon within the target gene. Typically, the floxed exon does not contain a unit number of codons so that the Cre-mediated recombination event yields a frameshift and a null allele. Documenting recombination within the genomic DNA is often regarded as sufficient proof of a frameshift, and the analysis of transcripts is neglected. We evaluated a previously reported conditional knockout allele for the beta-subunit of protein farnesyltransferase. The recombination event in that allele-the excision of exon 3-was predicted to yield a frameshift. However, following the excision of exon 3, exon 4 was skipped by the mRNA splicing machinery, and the predominant transcript from the mutant allele lacked exon 3 and exon 4 sequences. The "Deltaexon 3-4 transcript" does not contain a frameshift but rather is predicted to encode a protein with a short in-frame deletion. This represents a significant concern when studying an enzyme, since an enzyme with partial function could lead to erroneous conclusions. With thousands of new conditional knockout alleles under construction within mouse mutagenesis consortiums, the protein farnesyltransferase allele holds an important lesson-to characterize knockout alleles at both the DNA and RNA levels.
构建条件性敲除等位基因的常用策略是在目标基因内的一个5'外显子两侧“flox”（loxP位点）。通常，被floxed的外显子不包含整组密码子，以便Cre介导的重组事件产生移码突变和无效等位基因。记录基因组DNA内的重组通常被视为移码突变的充分证据，而转录本分析则被忽视。我们评估了先前报道的蛋白质法尼基转移酶β亚基的条件性敲除等位基因。该等位基因中的重组事件——外显子3的切除——预计会产生移码突变。然而，在外显子3切除后，mRNA剪接机制跳过了外显子4，突变等位基因的主要转录本缺少外显子3和外显子4序列。“缺失外显子3 - 4转录本”不包含移码突变，而是预计编码一种具有短框内缺失的蛋白质。在研究一种酶时，这是一个重大问题，因为具有部分功能的酶可能会导致错误的结论。在小鼠诱变联合体中正在构建数千个新的条件性敲除等位基因，蛋白质法尼基转移酶等位基因提供了一个重要教训——在DNA和RNA水平上对敲除等位基因进行表征。
