[Construction and functional identification of eukaryotic expression vector carrying Sprague-Dawley rat MSX-2 gene].
OBJECTIVE
To construct a high effective eukaryotic expressing plasmid PcDNA 3.1-MSX-2 encoding Sprague-Dawley rat MSX-2 gene for the further study of MSX-2 gene function.


METHODS
The full length SD rat MSX-2 gene was amplified by PCR, and the full length DNA was inserted in the PMD1 8-T vector. It was isolated by restriction enzyme digest with BamHI and Xhol, then ligated into the cloning site of the PcDNA3.1 expression plasmid. The positive recombinant was identified by PCR analysis, restriction endonudease analysis and sequence analysis. Expression of RNA and protein was detected by RT-PCR and Western blot analysis in PcDNA3.1-MSX-2 transfected HEK293 cells.


RESULTS
Sequence analysis and restriction endonudease analysis of PcDNA3.1-MSX-2 demonstrated that the position and size of MSX-2 cDNA insertion were consistent with the design. RT-PCR and Western blot analysis showed specific expression of mRNA and protein of MSX-2 in the transfected HEK293 cells.


CONCLUSIONS
The high effective eukaryotic expression plasmid PcDNA3.1-MSX-2 encoding Sprague-Dawley Rat MSX-2 gene which is related to craniofacial development can be successfully reconstructed. It may serve as the basis for the further study of MSX-2 gene function.
Yang
Xian-Xian
XX
Department of Plastic and Reconstructive Surgery, Shanghai 9th People' s Hospital, Shanghai 200011, China.
Zhang
Mei
M
Yan
Zhao-Wen
ZW
Zhang
Ru-Hong
RH
Mu
Xiong-Zheng
XZ
D000818:Animals; D003001:Cloning, Molecular; D015870:Gene Expression; D005801:Genes, Homeobox; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000235:genetics*; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D017422:Sequence Analysis, DNA
D018398:Homeodomain Proteins; C084119:MSX2 protein; D012333:RNA, Messenger