人t-PA突变体cDNA打靶敲入小鼠ES细胞β-酪蛋白位点的研究

Study on a Human Tissue-type Plasminogen Activator Mutant cDNA Knocked-in the β-casein Gene Site of Murine ES Cells

应用克隆的基因组序列作为同源臂 ,构建了小鼠 β 酪蛋白基因定位敲入打靶载体。短臂长 2 7kb ,包括小鼠 β 酪蛋白基因 5′端侧翼区、第 1外显子、第 1内含子、部分第 2外显子。长臂包括小鼠 β 酪蛋白基因部分第 2内含子、第 3～ 7外显子、第 3～ 6内含子、部分第 7内含子 ,长 3 4kb。人t PA突变体cDNA在第 2外显子中与小鼠 β 酪蛋白信号肽序列融合。正筛选标记neo放置在 β 酪蛋白基因第 2内含子中部 ,负筛选标记tk位于短臂外侧。ES细胞株TC 1在滋养层上培养扩增。处理ES细胞使其密度达到 2× 10 7个 /mL后 ,将 4 5 μg线性化的打靶载体DNA与 1mL细胞混匀后电击。转染的细胞在含G4 18和Gancyclovir的选择培养基中培养 ,7d后挑取 192个抗性克隆 ,扩增、提取基因组DNA ,EcoRⅠ酶切后 ,用打靶载体 5′端内侧探针进行Southern杂交 ,野生型出现 9 8kb ,而中靶的 β 酪蛋白基因由于敲入的人t PA突变体携带一个EcoRⅠ位点 ,中靶等位基因出现 6 6kb。从 78株ES细胞株中获得 1株发生正确同源重组的中靶ES细胞。

A murine β-casein gene targeting vector was constructed using the cloned genomic sequence.The short arm was 2.7 kb including mouse β-casein gene 5′ flanking sequence,exon1,intron1 and partial exon2.The long arm is a 3.4 kb fragment including partial intron2,exon3～7,intron3～6 and partial intron7.The human t-PA mutant cDNA was subcloned in the exon2 and fused with the mice β-casein signal peptide sequence.The positive selective marker neo^r was placed in the middle of intron2.A tk negative selective marker was just outside the short arm.TC-1 ES cells were cultured and amplified on G418 resistant feeder layer.The linearized targeting construct DNAs of 45 μg were introduced into 2×10~7 ES cells by electroporation.Totally 192 ES clones were picked up after cultured in G418 and Gancyclovir for 7 days.The colonies were amplified and subjected to genomic DNA preparation.The genomic DNAs were digested with EcoRⅠ and used for Southern blot analysis.A probe inside the 5′ homologous arm was used for hybridization.A 9.8 kb band was found in wild type,but the band was shift down from 9.8 kb to 6.6 kb in the β-casein gene targeted allele because a new EcoRⅠ site was introduced into the exon2 along with the human t-PA mutant gene.There were 9.8 kb and 6.6 kb bands in targeted ES cells.One clone of targeted ES cells with correct homologous recombination events was obtained among 78 analyzed clones.It lays foundation for gene targeted mice making.