摘要
为了利用ES细胞基因打靶技术建立人血友病B的转基因小鼠模型，用小鼠FIX基因cDNA为探针，筛选129Sv小鼠的基因组DNA噬菌体文库，从2×105个噬斑中得到5个独立的阳性噬菌体克隆．其中一个（C16）克隆含有约22kb插入片段，经Dotblotting，PCR，部分DNA序列测定和双酶组合分析等，完成了酶切图谱分析和外显子的定位，发现小鼠FIX基因组DNA的结构与人FIX基因组存在区别，主要表现在内含子的大小和序列上．分别选用含有第1，2个外显子的4．5kbBamHⅠ／XbaⅠ片段和含有第7，8外显子的1．8kbBamHⅠ／HindⅢ片段作为基因打靶置换型载体中长短同源臂，定向克隆人通用置换型载体骨架pSSC－9两个多克隆位点中，构建得到预计可以造成含有第3～6个外显子的大片段缺失突变的置换型载体（pMFIXDEL）．用载体两侧的SfiⅠ稀有酶切位点，将pMFIXDEL线性化．通过电穿孔法将25μg载体DNA转人约107个ES细胞中，分成GANC－／G418＋和GANC＋／G418－两组进行10d药物选择（药物浓度分别是GANC：2μmolβ／L，G418：200U／ml，选择从电穿孔后24h开始）．挑取药物抗性细胞，扩大培养后，用PCR和基因组Southern杂交进行药物抗性细胞的分子鉴定，结果获得4个（两个药物选择组分别是2／72，2／7）发生了预计的同源重组的ES细胞克隆，?
In order to establish coagulation factor Ⅸ gene knockout mouse for human haemophilia B, 2×105 phage plaques of 129Sv mouse genomic library were screened with plaque in situ hybridization screening with 32P-labeled cDNA probe 5 positive clones were finally obtained. The insert of one of the clones (C16),about 22 kb in length, was confirmed to contain the genomic DNA of mouse FIX gene with dot blottging, PCR and partial sequencing. The junction sites of 5' flanking sequence/exonl, exonl/intronl, intronl /exon2, exon7/nitron7/exon8, exons/3' flanking sequence, together with the whole length of nitron7 were sequenced. Restriction mapping and exon localization was performed. Compacred with its human counterpart,the genomic structure of mouse FIX gene was quite different in the size and sequence of nitrons, though the exon/intron organization type was the same.To disrupt the mouse FIX gene grossly,a replacement vector (pMFIXDEL) was constructed by applied 4. 5 kb BamH Ⅰ/Xba Ⅰ fragment (containing exon 1 and exon2)and 1. 8 kb (containing exon7 and exon8) of mouse FIX genomic DNA as long and short homologous arms, respectlvely, and inserted them into Xho Ⅰ /Sal Ⅰ and BamH Ⅰ /Hind Ⅲ site of two multiple cloning site in pSSC-9 plasmld. The linearized vector DNA were electroporated into R1 ES cells. After 10 days selection with G418 and Gancyclovir (GANC), 81 drug resistant clones were picked, expanded and molecular identified with PCR and genomic Southern blotting. 4 targeted clones were finally obtained. The mean homologous recopblnatlon frequency was 1. 66×10-6. For identification, PCR approach is sensitive and simple, but there was false-positive, while genomic Southern blotting is conceivable method to confirm the genomic structure after homologous recombination.
出处
《复旦学报（自然科学版）》
CAS
CSCD
北大核心
1998年第4期577-578,共2页
Journal of Fudan University：Natural Science
基金
国家自然科学基金
上海市生命科学研究中心研究基金
军队九五重点攻关项目
关键词
凝血因子Ⅸ
血友病
DNA
FIX基因组
coagulation factor Ⅸ
haemophilia B
gene targeting
embryonic stem cell
