Due to the shortage of human donors, treatment of type 1 diabets by transplantation therapy requires alternative wources. Human embryonic stem cells are ideal candidate for large-scale production of functional beta cells in vitro. However, currently available differentiation protokols only produce immature beta cells with poor glucose responsiveness. To optimize the differentiation protocok we at our labotatory aim to establish a hES reporter cell line for PTF1A, a key regulator of early pancreatic differentiation, using gene targeting by homologous recombination. When hES cells differentiate into PTF1A+ pancratic progenitor cells, the fluorescent reprter gene will be expressed from the endogenous promotor, enabling ur to follow the PTF1A expression in real time.
Using BAC recombineering techniques, we generated a PTF1A-GFP targeting vector. The targeting vector was then transfected into hES cells. Approximately 300 resistant clones were isolated by neomycin selection. PCR analysis of chromosomal DNA from these clones identified five clones containing the GFP reporter cassette incorporated into the PTF1A locus at the desired position. the efficiency of homologous recombination is 1.6%. These reporters are now used to facilitate directed differentiation of multipotent beta cell progenitors.
Last updated: June 1, 2011
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