Development of PCR Based Method for Gene Expression Analysis and Copy Number Estimation of Glycosylation Enzymes and Cloning of Gene ‘X’ and ‘Y’

Abstract

Specific PCR based methods for the quantitation of the all 5 genes were developed for the determination of the copy number of the integrated gene. The same method will also be used for mRNA expression analysis after incorporation of a Reverse transcription step. These methods will be used to quantitate the copy number of integrated glycosylation enzyme gene/s and later to quantitate the expression of the gene/s at the level of transcription. PCR for all the glycosylation enzyme genes has been optimized and these optimized conditions and methods would be further used to check expression of these genes in CHO cells transfected with the glycosylation gene(s) and their copy number in the CHO genome. The developed PCR based method is shighly sensitive and able to detect 10 fg of plasmid DNA which corresponds to 1235 copies of target gene. Real time PCR reaction resulted in sensitivity of 1 fg corresponding to 123 copies of target gene when 4 pM concentration of primers were used. Recombinant proteins (r-proteins) are gaining importance for the treatment and prevention of various human diseases. Different expression systems have been developed for the market-scale production of these proteins with mammalian, bacterial and fungal systems being the major ones. The genes to be cloned were glycosylation enzyme genes. More number of transformants were obtained on test ligation plates and will be further screened to select positive clones.