Creating Growth Hormone Resistance in Cells using a Hammerhead Ribozyme Approach
In an effort to create growth hormone (GH) resistance in mouse L cells and in mice, we have designed a ribozyme (RZ) directed against mouse GH receptor (GHR) mRNA. The ability of the RZ to specifically cleave GHR mRNA at the designated target site was verified by in vitro analysis. The ability of the RZ to cleave more than one substrate was also demonstrated in vitro. We have established several stable mouse L-cell lines expressing different amounts of mouse GHR. A metallothionein driven “mini-gene” containing the mouse GHR cDNA with added introns flanking exon 8a allowing for the production of both GHR and GH binding protein (GHBP) was used to create the cell lines. GH binding assays followed by Scatchard analysis revealed GHR numbers per cell ranging from ~50,000 to ~240,000. Western blot analysis using an antibody against phosphorylated tyrosines demonstrated a correlation between GHR number and STAT-5 phosphorylation. A linear relationship existed between receptor number and GH stimulated STAT-5 activation up to ~150,000 GHRs per cell, with maximal activation occurring at ~300,000. One of the GHR/BP cells lines that expressed ~170,000 GHRs per cell (E6 cells) was transfected with GHR-RZ coding DNA. Western blot analysis revealed a ~50% decrease in GHR abundance in RZ transfected cells. This decrease was confirmed by GH binding assays, which revealed the RZ transfected cells had ~81,000 GHRs per cell compared to ~170,000 without the RZ. This decrease in receptor levels was enough to greatly reduced E6 cell sensitivity to GH as shown by a decrease in GH-dependent STAT-5 activation, thus causing GH resistance in these cells. The same GHR-RZ was used to establish several transgenic mouse lines. Analysis of growth revealed no significant change between transgenic mice containing the RZ “mini-gene” as compared to non-transgenic littermates. Western blot analysis of GHR levels in the liver samples revealed that the RZ-DNA positive transgenic mice had similar GHR levels to that of control mice. Therefore, the GHR-RZ was successful at creating GH resistance in cultured cells but not in mice.
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:ribozyme growth hormone receptor resistance transgenic animals
Date of Publication:01/01/2001