Question

evaluating the outcome of transforming genetically modified e.Coli cells with human coagulant factor ix cdna'

Answer 1

blood disorder that result in the malfunctioning of the gene that codes for the expression of human factor IX proteins in the coagulation cascade thus leading to an imbalance in haemostatic regulation of blood clot in damage blood vessels. The primary aim of this study was to clone the coding sequence of human factor IX into a bacterial vector (pETBlue 2 plasmid) for transient expression of hFIX protein in E.coli cells. RNA extraction from HepG2 cells was carried out from which aliquots obtained were used for cDNA synthesis using M-MLV Reverse transcriptase enzyme. Synthesised cDNA sample was subjected to PCR analysis with different annealing temperatures (57⁰C, 55⁰C,52⁰ and 50⁰C) for amplification of hFIX coding sequence using primers designed from the coding sequence. Sucessful transformation of E.coli cells was observed through the blue and white screening analysis of colonies. Evidence for transcient expression of hFIX protein in E.coli could not be ascertain as at the time of this research.