SOUTHERN ILLINOIS UNIVERSITY
Grant: $9,063 - National Institutes of Health - Jun. 19, 2009
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Award Description: The research focus of my lab is identifying and characterizing the function of a recently discovered family of homeobox transcription factors, the Rhox genes. These genes are normally selectively expressed in reproductive tissues and are presumed to function in the development of the gonads and as regulators of fertility. Knockouts mice lacking the founding member of the family, Rhox5, exhibit reduced spermatogenic output due to increased death of germ cells, partially due to aberrant metabolism and regulation of insulins. The overall aim of my currently funded RO3 is to identify several novel putative cofactor proteins that interact with RHOX5 to regulate the Insulin2 promoter in Sertoli cells. Working exclusively with the aid of undergraduate researchers, the majority of the progress to date has been establishing the tools necessary to identify RHOX5 cofactors. For example, we have optimized two new anti-RHOX5 antibodies that are now commercially available from Imgenex and have a handle on their specificity (low crossreactivity with other recombinant RHOX proteins) and conditions for pull downs. We have prepared ?bait? expression constructs in a variety of different plasmid-based systems using full-length, as well as domain-specific (i.e. homeodomain, amino terminal deletions etc) proteins. As my students have helped me assemble the various items involved with the project, they have not really been able to appreciate the big picture of where the research is leading because they are required to work an hour here and there as their classes allow. My goal for the summer is to have them working full time in the lab and showing them that while their time spent developing tools are lab ?successes?, the real pay offs come from using them to generate data, something that I don?t think they appreciate yet. Something that I need to ramp up now that my lab is established here at SIU and the grace period has ended. I have two students that are versed in general molecular biology lab protocols and would like to hire another raw talent to begin the training process. The proposed summer research is the logical continuation of the experiments outlined in the original proposal. Specifically, using ?bait? molecules with different tagged strategies to purify cofactors that work in conjunction with RHOX5 to regulate the insulin promoter in Sertoli cells. RHOX5 Halotag labeled proteins will allow us to perform magnetic resin-based pulldowns, provide fluorescent labels of multiple colors for multiplex analyses, and direct IR dye labeling for real-time EMSA analyses.
Project Description: The first student concentrated on identifying new RHOX5 cofactors. They used a combination of Sertoli cell lines and testes extracts as source materials for the purification of the RHOX5 cofactor and helped establish the Rhox5-null mouse colony. The student that had been maintaining the animal colony graduated and moved on, so this was a key advance in laboratory training for this new student that will serve them well when they move to another position. Rhox5-null founder animals were transferred to SIU after a few setbacks in transit and breeding once they arrived. We are performing binding experiments using testis extracts lacking endogenous RHOX5 to reduce interference with the efficiency of the recombinant RHOX5 bait constructs. The second student focused on a secondary project that arose during the course of my proposed work that was not specifically part of my original specific aims. That is investigating the role of RHOX8 as a putative cofactor for RHOX5. We have performed a pull down with RHOX5 bait, in the presence of Ins2 promoter oligo fragments, and discovered that RHOX8 is present in the complex. The direct interaction between RHOX8 and the Ins2 promoter occurs in the absence of RHOX5, but RHOX8?s affinity for the promoter is much greater when RHOX5 is present in vitro. The concept that RHOX5 and RHOX8 work together on the Ins2 promoter to promote germ cell survival, and that in the absence of RHOX5, RHOX8 is capable enough to allow subfertility of Rhox5-null males has served as the basis of a new Rhox8-null NIH grant. We have developed two targeting strategies to knockout Rhox8. Constructs for siRNA knockdown of Rhox8 have been handed off to the transgenic core facility and we should have pups within a month or two. We recently discovered an error in our plan for the floxed conditional knockout so we are currently adjusting the construct before generating the mice. However, this should be underway by the end of October.
Jobs Summary: The HS student was introduced to reproductive physiology, molecular biology, and how genetic mouse models can be used to examine human maladies. These skills will be useful for future lab work or her pursuit of a MD degree. The undergraduate fellows expanded their knowledge and technique base and are now prepared for graduate school. Both plan to obtain MS degrees after their summer experience. (Total jobs reported: 1)
Project Status: More than 50% Completed
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