SETDB1 genomic DNA 를 표적하는 TALEN construct 제작 및 분석

국문 초록 (Abstract)

TALEN은 특정유전자를 표적 하여 knock-out 시킬 수 있는 새로운 개념의 유전자 클로닝 방법이다. TALEN 플라스미드에는 DNA binding 도메인과 Fok1 절단효소 기능이 융합되어 있기 때문에, genomic DNA 의 어느 부위라도 결합할 수 있고, 표적 염기서열을 절단하여 유전자 돌연변이를 유도할 수 있다. 본 연구에서 우리는 SETDB1 HMTase 유전자의 단백질 개시코돈과 프로모터 -25 upstream 부위를 표적 하는 두 종의 TALEN constructs 를 제작하였다. 이를 위하여 두 단계의 클로닝이 진행되었다. 첫 번째는 모듈벡터에서 pFUS배열벡터로 표적서열을 옮겨 콜로니 PCR을 통해 smear밴드와 Esp1 제한 효소를 이용하여 약 1 kb의 insert가 들어 있음을 확인하였다. 두 번째는 배열 벡터로부터 TALEN 발현벡터로 옮기는 과정을 진행하였으며, 염기서열분석을 통해 확인하였다. 그 결과 최초의 고안된 모듈벡터 서열들이 약 100 bp 간격으로 배열되어 있음을 확인하였다. 제작된 TALEN-DBEX2 construct는 transfection을 통해 SETDB1의 발현이 사라지는 것을 확인하였고, T7E1 분석을 통하여 표적부위에서 돌연변이가 발생하였음을 추정할 수 있었다. 한편, TALEN-DBPR25 transfection을 통하여서도 SETDB1의 발현이 감소하는 현상을 확인 하였다. DBEX2, DBPR25를 이입시킨 HeLa 세포에서 세포 형태가 길어지는 현상을 관찰할 수 있었다. 그러므로 단백질 개시코돈 또는 -25 upstream을 표적 하는 TALEN knock-out 방법은 SETDB1 유전자의 기능연구에 매우 유용하다고 사료된다.

다국어 초록 (Multilingual Abstract)

TALEN is a newly developed gene engineering method to knock out specific genes. It contains a DNA binding domain and a Fok1 nuclease domain in the TALEN plasmid. Therefore, the engineered TALEN construct can bind to any region of genomic DNA and cut the target nucleotide, thereby inducing mutation. In this study, we constructed two TALEN constructs targeted to a protein initiation codon (DBEX2) or the 25th upstream region (DBPR25) to enable mRNA synthesis of SETDB1 HMTase. We performed the TALEN cloning in two steps. The first step was from module vectors to pFUS array vectors. We confirmed successful cloning with a colony PCR experiment and Esp31 restriction enzyme digestion, which resulted in a smear band and a 1 Kb insert band, respectively The second step of the cloning was from a pFUS array vector to a mammalian TALEN expression vector. The engineered TALEN construct was sequenced with specific primers in an expression vector. As expected, a specific array from the module vectors was shown in the sequencing analysis. The specific module sequences were regularly arrayed in every 100 bp, and SETDB1 expression totally disappeared in the TALEN-DBEX2 transfection. PCR amplification targeting of DBEX2 was performed, and the PCR product was digested with a T7E1 restriction enzyme. The expression of SETDB1 was down-regulated in the TALEN-DBPR25 transfection. Morphological changes were also observed in the two TALEN constructs with transfected HeLa cells. These results suggest that the engineered TALEN constructs in two strategic approaches are very useful to knock-out of the SETDB1 gene and to study gene function.

목차 (Table of Contents)

• 서론
• 재료 및 방법
• 결과
• 고찰
• References
• 초록

참고문헌 (Reference)

1 Noh, H. J., "p53 down-regulates SETDB1 gene expression during paclitaxel induced-cell death" 1 : 43-48, 2014

2 Chen, K., "TALENs : customizable molecular DNA scissors for genome engineering of plants" 6 : 271-279, 2013

3 Aryan, A., "TALEN-based gene disruption in the dengue vector Aedes aegypti" 3 : e60082-, 2013

4 Schultz, D. C., "SETDB1 : a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins" 8 : 919-932, 2002

5 Sung, Y. H., "Knockout mice created by TALEN-mediated gene targeting" 1 : 23-24, 2013

6 Lavery, G. G., "Hexose-6-phosphate dehydrogenase knock-out mice lack 11 beta-hydroxysteroid dehydrogenase type 1-mediated glucocorticoid generation" 10 : 6546-6551, 2006

7 Galli-Taliadoros, L., "Gene knock-out technology : a methodological overview for the interested novice" 1 : 1-15, 1995

8 Holter, N. S., "Fundamental patterns underlying gene expression profiles : simplicity from complexity" 15 : 8409-8414, 2000

9 Tokumasu, D., "FAST-id system for enrichment of cells with TALEN-induced mutations and large deletions" 5 : 419-431, 2014

10 Yamaji, T., "Establishment of HeLa cell mutants deficient in sphingolipid-related genes using TALENs" 2 : e88124-, 2014

11 Lei, Y., "Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases(TALENs)" 43 : 17484-17489, 2012

12 Sommer, D., "Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases" 5 : 3045-, 2014

13 Cermak, T., "Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting" 39 (39): e82-, 2011

14 Carlson, D. F., "Efficient TALEN-mediated gene knockout in livestock" 43 : 17382-17387, 2012