RESUMO
BACKGROUND:Embryonic stem cells (ESCs) serve as a major cell source for smooth muscle cells,but the heterogeneity of cells derived from ESCs result in difficulty to obtain high purity smooth muscle cells.OBJECTIVE:To construct a double expression vector of puromycin resistance (pac) gene and enhanced green fluorescence protein (EGFP) gene driven by smooth muscle specific SM22α promoter (pSM22α-PAC-IRES2-EGFP),in addition,to detect its availability and specificity in ESCs.DESIGN,TIME AND SETTING:The observational experiment of gene level was performed at the Cardiovascular Institute,General Hospital of Shenyang Military Region from April 2007 to September 2008.MATERIALS:ESCs line R1 with number SCRC-1011TM was purchased from American ATCC Company.The pSM22α-EGFP vector was constructed by our laboratory.And the pIRES2-EGFP,pSM2C and pSuper.basic vectors were purchased from Invitrogen Company.METHODS:SM22α promoter was cloned from pSM22α-EGFP by polymerase chain reaction.CMV promoter of pIRES2-EGFP vector was replaced by SM22 promoter to establish pSM22α-IRES2-EGFP.Pac gene,excised from pSM2C by HindⅢ/Clal digestion,was sub-cloned into pSuper.basic to establish pSuper-PAC.After BgⅢ/Accl enzyme digestion of pSuper-PAC,pac gene fragment was obtained,which was further sub-cloned into pSM22α-IRES2-EGFP to produce pSM22α-PAC-IRES2-EGFP.ESCs were transfected with pSM22α-PAC-IRES2-EGFP using lipofectamine.Positive clones were selected by G418 and induced to differentiate and further identified by amplification of pac gene by RT-PCR.Differentiated cells were immunostained by SM α-actin,and expression of SM α-actin and EGFP was observed simultaneously under fluorescence microscope.MAIN OUTCOME MEASURES:Sequencing result of pSM22α-PAC-IRES2-EGFP;Amplification of pac gene;EGFP expression;as well as SM α-actin immunostaining.RESULTS:Three segments of 261 bp,664 bp,and 5000 bp were obtained by HindⅢ/Clal digestion,which was coincident with expectation,and the sequencing results showed that pSM22α-PAC-IRES2-EGFP vector was successfully constructed.Amplification of pac gene identified 4 ESCs clones successfully transfected.After induction of differentiation,partial portion of differentiated cells expressed EGFP,accompanied by positively stained by SM α-actin antibody.CONCLUSION:pSM22α-PAC-IRES2-EGFP vector was successfully constructed.ESCs clones transfected with this vector expressed pac gene and EGFP gene,and the expression of EGFP is smooth muscle specific.
