Erin and Di to present at Student Seminar Thursday (17.Sept) at noon in 2166 MERF
Background for Erin’s talk
Integrating vector systems are under current investigation as gene transfer vehicles in treatments for diseases such as cystic fibrosis (OMIM). Integrating vectors such as retrovirus, lentivirus, and DNA transposons are beneficial in achieving long-term expression of therapeutic genes. However, vector integration carries the potential for insertional mutagenesis, resulting in aberrant expression of host genes. Thus, it would be beneficial to develop vectors that target integration to a specific genomic region. This work aims to direct the DNA transposon vector piggyBAC to a specific locus in the genome. This strategy attempts to achieve site-directed integration through interaction of the DNA binding domain-modified transposase gene with its target in a human cell line. We have modified a codon-optimized piggyBAC transposase with the bacterial LacI DNA binding domain in an N-terminal fusion construct. Transposition assays showed that the modified transposase is active in human cells. Excision assays indicated that the modified transposase excises precisely. Moreover, we recovered intact transposase-genomic junctions from cells in which the modified transposase was delivered to catalyze transgene integration. These data indicate that in addition to precise excision from the transposon plasmid, the modified transposase can carry out correct integration reactions. Taken together, these results support the hypothesis that a piggyBAC transposase modified with a DNA binding domain can direct integration to a specific site in the genome. We are currently testing this in a proof-of-principle experiment in which we used a LacI-modified transposase to direct integration in a human cell line containing the target LacO DNA binding sites stably integrated at a single locus. Site-directed integration is an important step in developing safe and effective integrating vectors for therapeutic gene transfer applications.
Background for Di’s talk
Hypertension is defined as systolic blood pressure (SBP) over 140 mmHg and/or diastolic blood pressure (DBP) over 90 mmHg. It is an established risk factor for a range of diseases including obesity, diabetes, stroke and renal defects. Renin-Angiotensin System (RAS) plays an essential role in blood pressure regulation. Within this system, Renin (OMIM) is the rate-limiting enzyme in generating downstream effector angiotensin peptides. Renin is conventionally recognized as a secreted enzyme from renal JG cells. However, accumulating evidence support the existence and function of a novel Renin isoform that is retained intracellularly. We hypothesis that intracellular renin (icRen) plays a discrete role from its secreted counterpart (sRen), especially in the brain, to fine tune heart rate and blood pressure control via the central nervous system. I have completed a sRen-specific knockout mouse model during my PhD training. They exhibited lower blood pressure and renal atrophy due to the lack of sRen that is abundant in the kidney. This implicates that sRen is a vital isoform in the kidney and preservation of icRen mainly in the brain is not sufficient to rescue lethality.
Posted on September 14, 2009, in Student Seminar and tagged cystic fibrosis, Di Z., DNA transposon vector, Erin B., gene therapy, Hypertension, piggyBAC, Renin, Renin-Angiotensin System. Bookmark the permalink. Leave a comment.