論文名稱:

建立小鼠 tid1 基因之條件式基因剔除模式

 

Establishment of conditional knock out mouse model for

 

tid1, a putative tumor suppressor gene.

研究生:

莫居凡  Chu-Fan Mo

指導教授:

羅正汎  Jeng-Fan Lo

        學位類別:

碩士

        學校名稱:

國立陽明大學

系所名稱:

口腔生物研究所

            學號:

39417005

          學年度:

95

          語文別:

中文

          出版年:

96

關鍵字:

條件式基因剔除载體  conditional knock out targeting vector

 

基因標的  gene targeting

 

同質重組  homologous recombination

 

基因重組工程  recombineering

 

南方墨點法  Southern blot hybridization

 

  Tid1

全文說明:

電子全文

        論文頁數:

101

摘要:

  Tid1 蛋白為果蠅抑癌器 Tid56 在哺乳類的同源蛋白。Tid1 會藉由其Dna J domain heat shock protein 70 (Hsp70)此種chaperone 交互作用,進而參與蛋白複合體的合成與組裝。近年來一些研究證據顯示,Tid1是一個細胞內訊息傳遞路徑的調節者,與細胞存活、細胞增生,以及由壓力所誘發的許多細胞生理反應有關;另外一些細胞相關實驗則顯示,Tid1 對細胞而言有可能行使抑癌器的功能。然而至目前為止,由於欠缺一個良好的系統去觀察 Tid1 在生物體中參與癌化過程的作用機制,因此其是否真正在生物體中扮演抑癌器的角色,目前仍不清楚。

 

  本論文主要目的即是建構小鼠 tid1 基因的條件式基因剔除載體,

 

進而以同質交換(homologous recombination)得到正確的小鼠胚胎幹細胞突變株,並期望日後以此突變株建立起實驗小鼠系統,用於將來能更進一步去了解 Tid1 的生理功能及其參與癌化過程的作用機制。我們所利用的是ㄧ改良式高效率 recombineering 法,在特殊的大腸菌株中建構出 tid1 基因的條件式基因剔除載體,計畫剔除的區域為 tid1 基因的promoter exon 2,長約 5.8 kb,並使剔除之後的序列產生框架轉移突變(frame shift mutation),造成停止密碼(stop codon)提早出現,因而無法表現出完整的Tid1 蛋白。建構出此載體後以適當限制酶線性化,送入小鼠 129S6/SvEv-Tc1 品系的胚胎幹細胞進行同質重組,先經G418 ganciclovir 作初步篩選後,挑取 165 株具抗藥性幹細胞並抽取其基因體 DNA,再以南方墨點法確認 3’ 端及 5’ 端同質重組的正確性,結果共得到 1 3’ 端及 5’ 端皆有發生同質重組的胚胎幹細胞株。

 

  Tid1 is the mammalian counterpart of the Drosophila tumor suppressor Tid56 and is also a DnaJ protein containing a conserved J domain through which it interacts with the heat shock protein 70 (Hsp70) family of chaperone proteins. Several reports have indicated that Tid1 functions as a

 

regulator in intracellular signaling pathways related to cell survival, senescence, and stress-induced cellular responses. Other reports raised the interesting possibility that it may exert tumor suppressor activity through

 

cell-based assays. However, the biological function of Tid1 and how it acts as a tumor suppressor in vivo remain unclear because of lack of an instrumental in vivo analysis system.

 

  This research is focused on constructing a conditional knockout (CKO) targeting vector for mouse tid1 gene. We have employed a highly efficient recombineering-based method to make a tid1 CKO targeting vector in a specific Escherichia coli strain. The deleted region of tid1 gene is from putative promoter to exon 2, spanning approximate 5.8 kb genomic sequences, to not only disrupt the expression of tid1 transcript but also to cause a frameshift mutation with the consequence of early appearance of stop codon to generate null mutation. The constructed CKO targeting vector was subsequently linearized with restriction enzymes and electroporated into 129S6/SvEv-Tc1 ES cells to allow homologous recombination. The transformants were selected for their G418 and Ganciclovir (Ganc) resistance. Following electroporation, we screened 165 G418/Ganc resistant clones for correctly recombined clones by using Southern blot hybridization. We have up to now successfully obtained one clone of targeted ES cells with correct 5’ and 3' homologous recombination.