|
KMID : 0613820140240121269
|
|
Journal of Life Science
2014 Volume.24 No. 12 p.1269 ~ p.1275
|
|
|
TALEN Constructs and Validation for Targeting of SETDB1 Genomic DNA
|
|
Noh Hee-Jung
Kang Yoon-Sung
Kim Keun-Cheol
|
|
|
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.
|
|
|
KEYWORD
|
|
|
DBEX2, DBPR25, knock-out, SETDB1, TALEN
|
|
|
FullTexts / Linksout information
|
|
|
|
|
|
Listed journal information
|
|
|
|