Bautista D, Shulman M J
Department of Immunology, University of Toronto, Canada.
J Immunol. 1993 Aug 15;151(4):1950-8.
The traditional method of site-specific mutagenesis is to introduce predetermined mutations into an expression vector, which is then transferred to cells so that the relevant gene product and the effects of the mutations can be measured. A problem with this approach is that the expression of the transferred genes varies from transformant to transformant, presumably because the number of vector copies and the site of chromosomal integration vary among transformants. While it should be possible to avoid this variability by mutagenizing the chromosomal gene itself, the labor involved in introducing predetermined mutations by homologous recombination with a mutagenized vector is usually so intense that this has not been the favored method. We describe here a system for introducing mutations into the IgH locus of hybridoma cells. This system greatly reduces the labor that would usually be required to identify and recover the rare recombinants. This is a two-step, so-called "hit-and-run," method, whereby mutations are first introduced into the chromosomal locus by targeted vector integration, after which the vector is excised so as to leave the mutation in the chromosomal target. The first step employs an enhancer trap vector bearing an enhancerless gpt gene; using this vector the frequency of randomly inserted transformants which grow in mycophenolic acid containing selective medium is so low that approximately 25% of the selected transformants have integrated the vector into the IgH locus by homologous recombination. Properly targeted transformants can then be used to derive secondary recombinants that have excised the vector and thus become gpt-. This second step which involves selection of gpt- cells by their resistance to 6-thioxanthine is also efficient, in that approximately 75% of the treated cells have excised the gpt gene by homologous recombination. Overall the labor involved in mutagenizing the chromosomal locus is not much more than is needed to produce the traditional transformants expressing a mutagenized transferred gene.
传统的位点特异性诱变方法是将预先确定的突变引入表达载体，然后将其转入细胞，以便能够检测相关基因产物和突变的影响。这种方法存在一个问题，即转入基因的表达在不同转化体之间存在差异，推测这是因为载体拷贝数和染色体整合位点在不同转化体之间有所不同。虽然通过诱变染色体基因本身有可能避免这种变异性，但通过与诱变载体进行同源重组引入预先确定的突变所涉及的工作量通常非常大，以至于这种方法并不受青睐。我们在此描述一种用于在杂交瘤细胞的免疫球蛋白重链（IgH）基因座中引入突变的系统。该系统大大减少了鉴定和回收罕见重组体通常所需的工作量。这是一种两步的所谓“打了就跑”方法，首先通过靶向载体整合将突变引入染色体基因座，之后切除载体，从而使突变留在染色体靶点中。第一步使用携带无增强子gpt基因的增强子捕获载体；使用该载体，在含有霉酚酸的选择培养基中生长的随机插入转化体的频率非常低，以至于大约25%的选择转化体通过同源重组将载体整合到了IgH基因座中。然后可以使用正确靶向的转化体来获得已切除载体并因此变为gpt-的二级重组体。涉及通过对6-硫代黄嘌呤的抗性选择gpt-细胞的第二步也很有效，因为大约75%的处理细胞通过同源重组切除了gpt基因。总体而言，诱变染色体基因座所涉及的工作量并不比产生表达诱变转入基因的传统转化体所需的工作量多多少。
