My work in the Bowen-Pope lab involves two separate projects: a primary focus on PTP-RQ "RQ" and a secondary focus on PDGFb.
RQ is a receptor-like PTPase that was discovered in a rat renal injury model. RQ protein is expressed by rat renal mesangial cells, human renal podocytes, and mouse inner ear hair cells. Kiphos has multiple transcripts. The largest transcript (7.5 kb) encodes for a the receptor-like PTPase form of RQ. The smallest transcript (1.8 kb) encodes for the "truncation-deletion" intracellular variant form of RQ that has the catalytic domain but does not contain the transmembrane nor extracellular domains and cannot encode a receptor PTPase. A "knock-out" mouse was made where the transmembrane region was replaced such that a receptor-like RQ cannot be expressed. Subsequently it was determined that the 1.8 kb transcript is derived from an alternate promotor downstream of the transmembrane domain; so a second RQ "knock-out" (KO) mouse is being developed where both the receptor-like and intracellular variants of RQ with be eliminated.
We have not yet identified any histolopathologic or physiolotic phenotype in the transmembrane homozygote KO mice that cannot make the receptor-like form of RQ except for the cochlear hair cells. One of our collaborators has found that there is clear structural abnormalities in the hair bundle by ultrastructural examination. There is a subsequento loss of hair cells and the mice are deaf at a very young age.
In the summer of 2001 we had germline transmission in the second line of catylic KO mice. Recently the F1 heterozygotes were crossed and genotyping and histologic characterization of their pups are in progress.
The majority of my work has centered on determining the histologic and/or physiologic phenotype in the different lines of RQ mice. In addition, immunohistochemical (IHC) studies are in process to determine the cellular expression of RQ protein in human, nonhuman primate, and mouse tissues. As a component of this, portions of my work involve characterization of peptide antibodies to the C-terminal end of RQ. The antibody studies have shown that we can detect the receptor-like form of RQ protein in the sensory hair cells of the inner ear of mice. Determination of other cells that express RQ protien in specific tissues are in progress.
Additions studies to determine the role of RQ in injury are being planned.
The secondary project is centered on stellate (Ito) cells in the liver and the role of PDGFb in carbon tetrachloride (CCl4) induced hepatic injury. Mice lacking PDGFbdie during embryogenesis. In order to study the role of PDGFb, a chimeric model is utilized where 8 cell embryos from mice carrying the human globin gene (cellular marker) and wild type, heterozygous, or null for PDGFb are fused with an 8 cell embryo from a different line of mice. These "chimeric" mice are then injected with CCl4 and euthanized and necropsied at different time points. Stellate cells stain positive by desmin IHC. Cells from the PDGFbmice can be identified by in situ hybridization (ISH) to the human beta globin gene. Currently all the animal studies are completed and the tissues collected, processed and embedded. The first phase verifying stellate cell proliferation after hepatic injury by CCl4 is completed. The individual protocols for the DNA ISH and desmin staining by IHC both work individually. Dual staining has been accomplished but is in the process of being refined for optimum cell counting to complete the quantitative analysis of the response of stellate cells containing or lacking PDGF?in CCl4 induce hepatic injury. This project is anticipated being completed before this spring.