Shulman M J, Nissen L, Collins C
Department of Immunology, University of Toronto, Canada.
Mol Cell Biol. 1990 Sep;10(9):4466-72. doi: 10.1128/mcb.10.9.4466-4472.1990.
Mutant hybridoma-myeloma cell lines that are defective in immunoglobulin production are expected to be useful for defining the molecular requirements of immunoglobulin gene expression. The analysis of such mutants would be greatly facilitated if they could be mapped by marker rescue, i.e., by identifying the segments of wild-type DNA that can restore the normal phenotype by homologous recombination with the mutant chromosomal immunoglobulin gene. To assess the feasibility of this type of mapping, we have measured the efficiency with which fragments of wild-type DNA recombine with a mutant hybridoma immunoglobulin gene and restore normal immunoglobulin production. We found that most if not all recombinants were detectable 2 days after DNA transfer and that the frequency of gene restoration increased with increasing length of the transferred mu gene fragments, between 1.2 and 9.5 kilobases. These results indicate that the available technology should be adequate to map mutations in the mu gene to within approximately 1 kilobase.
预计在免疫球蛋白产生方面存在缺陷的突变杂交瘤 - 骨髓瘤细胞系可用于确定免疫球蛋白基因表达的分子要求。如果能通过标记拯救对这类突变体进行定位，即通过鉴定野生型DNA片段，这些片段可通过与突变染色体免疫球蛋白基因的同源重组恢复正常表型，那么对这类突变体的分析将得到极大促进。为评估这种定位类型的可行性，我们测定了野生型DNA片段与突变杂交瘤免疫球蛋白基因重组并恢复正常免疫球蛋白产生的效率。我们发现，大多数（如果不是全部）重组体在DNA转移后2天即可检测到，并且基因恢复频率随着转移的μ基因片段长度增加而增加，片段长度在1.2至9.5千碱基之间。这些结果表明，现有技术应足以将μ基因中的突变定位到约1千碱基范围内。
