Directed Stem Cell Differentiation
M. Ian Phillips, in collaboration with Daniela Castanotto and John Rossi at City of Hope, Yao Liang Tang at the University of Cincinatti, and Edilamar de Oliveira in San Paoulo University, Brazil
Cardiac ischemia frequently results in irreversible damage to cardiomyocytes, and eventually may lead to heart failure. A potential method to remedy myocardial damage is cell replacement therapy. Adult cardiac stem cells (CSCs) are multipotent cells residing in the heart and hold promise as a means to facilitate cardiac tissue repair. One of the major problems of stem cell therapy however is achieving appropriate differentiation – in this case transformation of CSCs to beating cardiomyocytes. We have started to use inhibition of microRNAs as a means to guide CSC differentiation into cardiomyocytes. Micro RNAs are small non- coding RNAs that target the mRNA of coding genes, and suppress protein translation. By inhibiting the micro RNA, this suppression is released and specific proteins are generated that trigger the CSC to differentiate.
Gene Vector Control of Bleeding
M. Ian Phillips
In many situations such as combat injury, surgery, rare bleeding diseases and cerebral stoke, hemorrhage needs to be stopped to prevent death. Our lab has developed an automatic gene vector hemostat based on genetic engineering and testing in human endothelial cells. The vector responds to the oxgyen change in injured tissues, and releases clotting factors locally in small enough amounts to stop bleeding, but unlikely to cause thrombosis. We collaborate with the US Army Surgical Institute (San Antonio, Texas) and the University of South Florida (Tampa) for use in battlefield combat injuries and with the neurology department of UC San Diego for the stroke studies.
Expression Strain Optimization
This project involves the construction of P. pastoris strains that are deleted for genes whose products are deleterious to recombinant protein production (e.g., proteases) and/or overexpress genes whose products may aid in recombinant protein synthesis (e.g., chaperones, protein disulfide isomerase). The DNA sequence of the genome of P. pastoris has been determined. Because P. pastoris is related to other budding yeasts, especially the well-known baker’s yeast Saccharomyces cerevisiae, the identity of most P. pastoris genes can be inferred from comparison to other yeast genes, which facilitates the production of modified strains that can produce higher yields of functional recombinant proteins.
Construction of Recombinant Protein Producing Strains
At present, Dr. Cregg’s laboratory is working with several companies on developing new expression strains for recombinant proteins of commercial interest. One is a phytase enzyme to be used as a nutritional additive in animal feeds. Others include several galactosidases, which help degrade lactose, a sugar that many animals and humans have difficulty digesting, and lactoferrin, which will be used as an anti-microbial agent.
Improvements to the P.pastoris Expression System
Three projects are underway to improve the overall abilities of the P. pastoris expression system. The first involves a search for improved promoters to drive foreign gene expression. DNA microarray hybridization have been performed using labeled P. pastoris RNAs taken from cells growing on different carbon sources. These have been searched for promoters that synthesize the highest levels of RNA. The promoters will be cloned and inserted into a test vector to confirm their suitability for foreign gene expression and then incorporated into our standard expression vectors for use. The second project involves testing new secretion signal sequences for their efficiency in secreting foreign proteins from P. pastoris. The third project involves the optimization of our DNA electroporation transformation protocol to improve the numbers of expression strains we can generate per ug of vector DNA.