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研究生:黃俊凱
研究生(外文):Chun-Kai Huang
論文名稱:CDK12, CDK13, Cyclin L1及Cyclin L2表現分析以及CDK12有條件基因剔除小鼠之建置
論文名稱(外文):Expression patterns of CDK12, CDK13, Cyclin L1, and Cyclin L2 and Generation of CDK12 Conditional Knockout Mice
指導教授:范明基
指導教授(外文):Ming-Ji Fann
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學暨基因體科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:57
中文關鍵詞:CDK12基因有條件剔除
外文關鍵詞:CDK12conditional knockout
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CDK12 是一個細胞週期依賴性激酶,在其氨基末端具有絲胺酸/精胺酸豐富區塊,而在其碳基末端則具有磷酸激酶區塊。過去研究指出 CDK12 在大鼠 E14.5 (授精後 14.5 天)胚胎的腦、脊髓、心臟、肺、腸以及四肢等組織都有表現。同時 CDK12 也被報導會與 L 型細胞週期素共同調控 RNA 的選擇性剪接。CDK13是另一個類細胞分裂週期蛋白 2 號(CDC2L),它也被發現會與 L 型細胞週期素產生交互作用並且調控 RNA 的選擇性剪接。在本篇論文中,使用了原位雜交(in situ hybridization)的實驗方法來分析 CDK12、CDK13、L 型細胞週期素 1 號(cyclin L1)以及 L 型細胞週期素 2 號(cyclin L2)的基因表現。實驗結果顯示 cyclin L1 在小鼠 E8.5 到 E9.5 的胚胎中表現在除了心臟以外的其他所有細胞中,到了 E10.5 則只表現在四肢、腮弧、前腦以及耳泡等區域;cyclin L2 在小鼠 E8.5 到 E9.5 的胚胎中表現在除了心臟以外的其他所有細胞中;CDK12 在小鼠 E8.5 到 E10.5 的胚胎中表現在前腦的區域;而在 E8.5 到 E9.5 的小鼠胚胎中我們並沒有偵測到 CDK13 的基因表現。
在先前的其他研究中也指出,當我們在已分化的 P19 細胞株中抑制 CDK12 或是 CDK13 基因的表現量,會使得細胞的神經突起的生長也受到抑制。這意味著在已經分化的神經細胞中,CDK12 以及 CDK13 參與維持神經細胞突起生長過程的角色。因此我們希望可以在活體動物中研究 CDK12 的功能,特別是它在神經系統中的可能功能。我們想利用基因剔除的方法來達到這個目的。由於 CDK12 在胚胎發育時期普遍的表現在許多組織中,全身性的基因剔除可能導致胚胎時期的死亡,因此有條件的基因剔除會是一個比較適合的方法。在實驗中我們利用大腸桿菌(E.coli)中同源基因重組的系統來置備 CDK12 有條件基因剔除的轉殖載體。我設計在 CDK12 的基因體中插入兩個 loxP 位置,在 Cre 表現之下,兩個分別位於 2 號內含子以及 4 號內含子的 loxP 位置會產生同源重組進而將 3 號外顯子以及4 號外顯子剔除。這會使得蛋白質轉譯時出現編碼框移的現象,造成轉譯被提前終止而生成了一個被截斷且不具有磷酸激酶區域的蛋白質。因此在有 Cre 的狀況之下可以達到 CDK12 基因剔除的目的。
我已經得到了 CDK12 有條件基因剔除的轉殖載體並且使用在小鼠胚胎幹細胞基因標的,經由南方轉漬法篩選到正確標的的胚胎幹細胞株。成功標的得胚胎幹細胞株目前正利用在進行小鼠囊胚顯微注射以產生嵌合鼠。
CDK12 is a cyclin dependent kinase which has a N-terminal serine/arginine-rich domain (RS domain) and a C-terminal kinase domain. In a previous study, CDK12 was shown to express ubiquitously in rat E14.5 tissues, including brain, spinal cord, heart, lung, gut, and limb. CDK12 was also shown to regulate alternative splicing together with L-type cyclins. Another CDC2L (cell division cycle 2-like) protein, CDK13, also interacts with L-type cyclins and regulate alternative splicing. In this report, expression patterns of CDK12, CDK13, cyclin L1 and cyclin L2 in mouse embryo were analyzed by in situ hybridization. Results show that cyclin L1 expresses in almost all cells except heart in E8.5 ~ E9.5 embryo and expresses at limbs, branchial arches, forebrain and otic vesicle in E10.5 embryo, cyclin L2 expresses in almost all cells except heart in E8.5 ~ E9.5 embryo, CDK12 expresses in forebrain during E8.5 ~ E10.5, and CDK13 does not express in E8.5 ~ E9.5.
In an another previous study, knockdown of CDK12 or CDK13 in differentiated P19 cell decreases neurite outgrowth, suggesting that CDK12 and CDK13 play a role in maintaining the neurite outgrowth of differentiated neuronal cells. Thus, it will be interesting to reveal functions of CDK12 in vivo, especially in the nervous system. As direct CDK12 knockout may cause embryonic lethality, a CDK12 conditional knockout approach seems more appropriate. We had generated a conditional knockout construct by a recombination system in E.coli. The design was to introduce loxP sites in intron 2 and intron 4 of CDK12 genome, such that exon 3 and exon 4 will be deleted in the presence of Cre activity. Deletion of these two exon leads to a frame shift in the coding sequence and translation will be terminated prematurally and the truncated protein doesn’t contain the kinase domain. Under this design, we can knockout CDK12 in the present of Cre activity.
I had already generated the CDK12 conditional knockout construct for mouse embryonic stem cell (ES cell) targeting. Targeted ES cell clones were performed for blastocyst injection. Blastocyst injection is now progressing to generate chimera mice.
ABSTRACT (IN CHINESE) I
ABSTRACT III
TABLE OF CONTENTS V
INTRODUCTION 1
Conditional knockout
CDK12 and CDK13
Cyclin L1 and cyclin L2
The goals of the present study
MATERIAL AND METHODS 6
Animals and reagents
Bacterial strains
Vector construction
Southern blotting
In situ hybridization
RESULT 13
Construction of CDK12 conditional knockout targeting vector
Southern blot screening for targeted chromosome
Expression patterns of CDK12 and CDK13
Expression patterns of cyclin L1 and cyclin L2
DISCUSSION 19
The design for CDK12 conditional knockout
To obtain conditional knockout mice from chimera founder
Knockout CDK12 by crossing with Cre line mice
CDK13 conditional knockout mice need to be generated
CDK12 and CDK13 might play their functions at different developmental stages
REFERENCES 24
TABLES 27
FIGURES 30
SUPPLEMENTARY FIGURES 40
APPENDIX 49
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