Construction of mouse model of human diseases by gene targeting

我们以小鼠胚胎癌F9细胞和转甲状腺激素(TTR)基因为模型系统，构建了两种基因打靶载体。我们使用了F9细胞，因为它易于操作，具有与胚胎干细胞相似的特性，并使用了TTR基因，因为人类基因的突变会导致家族性淀粉样变性多发性神经病。插入突变载体：该载体第2外显子含有5.9kd的新霉素耐药基因(Neo)，3‘端含有HSV-tk基因。HSV-tk基因3‘端DNA尾长越大，GANC选择效率越高。非靶向克隆中保留的载体DNA的结构表明它们具有核外降解作用。用发夹状寡核苷酸封端的载体DNA提高了GANC选择的效率。一种引入细微突变的载体：该载体由携带第二外显子3碱基突变的TTR片段和一盒neo和HSV-tk基因组成，该片段在第二外显子上携带一个3碱基突变，导致与FAP患者相同的TTR中的单一氨基酸替换，以及一盒neo和HSV-tk基因，两侧是TTR基因第二内含子3kd的重复。在第一步中，从G418r克隆中筛选出载体DNA通过同源重组部分替换内源Ttr基因的克隆。在第二步中，从目标G418^r克隆中筛选出选择标记盒已被染色体内重组去除的GANC^R克隆。有趣的是，许多克隆携带HSV-tk基因启动子区域的甲基化。在载体DNA的5个随机整合体中有效地切割了选择标记盒，表明该步骤适用于多种小鼠基因。

We developed two kinds of gene targeting vectors using mouse embryonal carcinoma F9 cell and a transthyretin (ttr) gene as a model system. We used F9 cell, because it is easy to handle and has sinilar properties to embryonal stem cell, and used the ttr gene, because mutations of the human gene cause familial amyloidotic polyneuropathy.1. A vector to introduce insertional mutations : This vector consisted of a 5.9 kd ttr fragment carrying neomycin-resistance (neo) gene in the 2nd exon and a HSV-tk (herpes simplex virus thymidine kinase) gene at the 3' end. When the plasmid DNA tail at the 3' end of HSV-tk gene was longer, the efficiency of GANC selection was higher. Structues of the vector DNAs retained in non-targeted clones suggested their exonucleolytic degradation. Capping the ends of vector DNAs with hairpin-shaped oligonucleotides increased the efficiency of GANC selection.2. A vector to introduce subtle mutations : This vector consisted of the ttr fragment carrying a 3-base mutation in the 2nd exon that causes a single amino acid substitution in TTR identical to FAP patients, and a cassette of neo and HSV-tk genes flanked with a 3 kd duplication of the 2nd intron of ttr gene. In the 1st step, those clones, in which part of the endogenous ttr gene was replaced by vector DNA through homologous recombination, were selected from G418^r clones. In the 2nd step, GANC^r clones, in which the selection marker cassette had been excised by intrachromosomal recombination, were selected from the targeted G418^r clones. Interestingly, many GANC^r clones carried HSV-tk gene methylated at the promoter region. The selection marker cassette was efficiently excised in 5 random integrants of the vector DNA,indicating that this step is applicable to a wide variety of mouse genes.