Construction And Characterization Of ADAM10 Conditional Gene Targeting Vector

【Object】:Construction of ADAM10 conditional gene targeting vector is to lay the foundation of preparing the ADAM10 conditional knockout mice and to study the function of ADAM10 gene.【Material and Method】:Genomic DNA fragments surrounding exon 2 of ADAM10 gene were amplified from 129/SVJ mice by PCR.Partial fragments of which were sequenced.The 2.5kb short arm,4.9kb long arm and the exon2 were ligated to the targeting skeleton vector pL451,respectively,to form the intermediate vector pEFUD.The final targeting vector pEFUD-TK was constructed by ligating the HSV-TK gene fragment isolated from the pSSC9plasmid to the vector pEFUD.In order to understand whether the Neo and HSV-TK gene in the final construct play a normal role in vitor,the plasmid pEFUD-TK,pL451 and pSSC9 were separately transfected into human neuroblastoma cells (SH-SY5Y) by LiPide2000(control group:pSSC9,pL451 and zero;test group: circular pEFUD-TK and linear PEFUD-TK).【Result】:Restriction endonuclease digestion showed that the restriction mapping of long arm and short arm is identical with that of the reported sequences from 129/SVJ mouse.Sequencing results indicated that the determined sequences are nearly the same as those of the reported sequences(99.8%),especially,the exon2 sequence is completely identical with the reported.Exon 2,long arm and short arm were exactly inserted in pL451.The HSV-TK gene sequence from pSSC9 is also the same as the reported.The targeting construct vectors were finally completely.After the following plasmid and constructs were transfected into In SH-SY5Y cells by LP2000,small colonys were detected in test group pSSC9,pL451, circular pEFUD-TK and linear pEFUD-TK,while not in control group zero, by using G418(850ng/μl) screening of 10 days.Subsequently,small colonys were almost disappeared in group pSSC9,circular pEFUD-TK and linear pEFUD-TK,but surviveded and grew in group pL451,by using GCV(10ng/μl) screening for 72 hours after G418 selection.【Conclusion】:The ADAM10 conditional gene targeting vector(pEFUD-TK) was successfully constructed and identified.The Neo and HSV-TK gene in the final construct can perform a normal function in the cell.