Division of Developmental Genetics, Institute of Resource Development and Analysis, Kumamoto University, Honjo 2-2-1, Kumamoto, Japan.
Mamm Genome. 2013 Jun;24(5-6):228-39. doi: 10.1007/s00335-013-9452-4. Epub 2013 Apr 20.
The MSM/Ms strain is derived from the Japanese wild mouse Mus musculus molossinus and displays characteristics not observed in common laboratory strains. Functional genomic analyses using genetically engineered MSM/Ms mice will reveal novel phenotypes and gene functions/interactions. We previously reported the establishment of a germline-competent embryonic stem (ES) cell line, Mol/MSM-1, from the MSM/Ms strain. To analyze its usefulness for insertional mutagenesis, we performed gene-trapping using these cells. In the present study, we compared the gene-trap events between Mol/MSM-1 and a conventional ES cell line, KTPU8, derived from the F1 progeny of a C57BL/6 × CBA cross. We introduced a promoter-trap vector carrying the promoterless β-galactosidase/neomycin-resistance fusion gene into Mol/MSM-1 and KTPU8 cells, isolated clones, and identified the trapped genes by rapid amplification of cDNA 5'-ends (5'-RACE), inverse PCR, or plasmid rescue. Unexpectedly, the success rate of 5'-RACE in Mol/MSM trap clones was 47 %, lower than the 87 % observed in KTPU8 clones. Genomic analysis of the 5'-RACE-failed clones revealed that most had trapped ribosomal RNA gene regions. The percentage of ribosomal RNA region trap clones was 41 % in Mol/MSM-1 cells, but less than 10 % in KTPU8 cells. However, within the Mol/MSM-1 5'-RACE-successful clones, the trapping frequency of annotated genes, the chromosomal distribution of vector insertions, the frequency of integration into an intron around the start codon-containing exon, and the functional spectrum of trapped genes were comparable to those in KTPU8 cells. By selecting 5'-RACE-successful clones, it is possible to perform gene-trapping efficiently using Mol/MSM-1 ES cells and promoter-trap vectors.
MSM/Ms 品系源自日本野生鼠 Mus musculus molossinus，具有不同于常见实验室品系的特征。使用基因工程 MSM/Ms 小鼠进行功能基因组分析将揭示新的表型和基因功能/相互作用。我们之前报道了从 MSM/Ms 品系中建立的具有种系能力的胚胎干细胞（ES）细胞系 Mol/MSM-1。为了分析其用于插入诱变的有用性，我们使用这些细胞进行了基因捕获。在本研究中，我们比较了 Mol/MSM-1 和来自 C57BL/6×CBA 杂交 F1 后代的常规 ES 细胞系 KTPU8 的基因捕获事件。我们将携带无启动子β-半乳糖苷酶/新霉素抗性融合基因的启动子捕获载体引入 Mol/MSM-1 和 KTPU8 细胞中，分离克隆，并通过快速扩增 cDNA 5'-末端（5'-RACE）、反向 PCR 或质粒拯救鉴定捕获的基因。出乎意料的是，Mol/MSM 陷阱克隆中的 5'-RACE 成功率为 47%，低于 KTPU8 克隆中的 87%。5'-RACE 失败克隆的基因组分析表明，大多数克隆捕获了核糖体 RNA 基因区域。在 Mol/MSM-1 细胞中，核糖体 RNA 区域捕获克隆的百分比为 41%，而在 KTPU8 细胞中则低于 10%。然而，在 Mol/MSM-1 5'-RACE 成功克隆中，注释基因的捕获频率、载体插入的染色体分布、整合到起始密码子含exon 周围内含子中的频率以及捕获基因的功能谱与 KTPU8 细胞相当。通过选择 5'-RACE 成功克隆，可以使用 Mol/MSM-1 ES 细胞和启动子捕获载体有效地进行基因捕获。
