Construction and identification of a recombinant adenovirus vector expressing rat achaete-scute homology 1 / 中国组织工程研究

ABSTRACT

BACKGROUND:Previous studies have suggested that achaete-scute homology 1 (ASCL1) plays a key role in the neuronal commitment. Therefore, somatic cels may directly differentiate into neurons by gene transfection ofASCL1, which wil provide new therapeutic strategies for optic nerve regeneration. OBJECTIVE:To construct a recombinant adenovirus vector expressing ratASCL1 gene for further research ofASCL1 gene function. METHODS:The ratASCL1 gene and advenovirus shuttle plasmid (pYr-adshuttle-4) which contained enhanced green fluorescent protein (EGFP) reporter gene were cleaved by restriction endonucleaseXhoI andEcoR I. The target gene fragments were connected together to generate a recombinant plasmid pYr-ads-4-rat-ASCL1 and then transfected into E.coliDH5α. The plasmid was confirmed to be constructed as expectation by enzyme digestion and sequence reaction. The plasmid pYr-ads-4-rat-ASCL1 and pAd/PL-DEST were reconstructed by homologous recombination processes to obtain rat ASCL1 recombinant adenovirus vector. The plasmid pYrAd-rASCL1 was linearized byPac I and subsequently transfected into HEK293 cels for packaging and amplification. RatASCL1 gene in the recombinant adenoviruses were identified by PCR. Virus titer was determined by tissue culture infectious dose 50. Infection efficiency was monitored by EGFP expression. RESULTS AND CONCLUSION:Restriction endonuclease digestion and DNA sequencing showed that the recombinant adenovirus vector pAd-rat-ASCL1 was constructed correctly. The positive amplification bands of 862 bp could be seen in PCR analysis. The virus titer reached 2×1010 pfu/mL. Infection efficiency of recombinant adenovirus in HEK293 cels was more than 80%. The results indicate that the recombinant the adenovirus vector containingASCL1 with high titer and infection efficiency has been successfuly constructed, which can be helpful for further research of the function and clinical application ofASCL1 gene for optic nerve regeneration.