134 BRAHMA-RELATED-PROTEIN 1 OVEREXPRESSION IN PARTHENOGENETIC PORCINE EMBRYOS

Abstract

The chromatin remodeling protein Brahma-related-protein 1 (Brg1) has been implicated in several cellular processes that affect chromatin structure and transcription. Brg1 is a member of the SWI/SNF family, a large group of chromatin remodeling proteins, all of which have the characteristic of utilizing the energy from ATP hydrolysis to catalyze the repositioning of nucleosomes on DNA templates. Various members of this class of proteins are known to be differentially expressed throughout vertebrate development. We hypothesize that the balance of chromatin remodeling factors present in cleavage stage porcine embryos has an impact on embryonic developmental potential. As a first step to testing this hypothesis, we attempted to overexpress a wildtype version of Brg1 in cleavage stage porcine parthenogenic embryos. The Brg1 construct was a gift from Dr. Anthony Imbalzano (University of Massachusetts, Worster, MA). The Brg1 cassette ligated into the NheI site of pIRES2-eGFP, Clontech. This construct was sequenced to verify the orientation. Oocytes were matured in vitro in defined culture medium (TCM199 supplemented with 0.1% polyvinyl chloride, 10 ng/mL epidermal growth factor, 0.5 IU/mL LH, 0.5 IU/mL FSH, 1 mm cysteine) for 42-44 h at 39°C in 5% CO₂. Following in vitro maturation, oocytes were denuded and activated by electroporation and cultured in NCSU23 supplemented with BSA (4 ¼g/mL) at 39°C in 5% CO₂, 5% O₂. For microinjection, activated oocytes were divided into 3 treatment groups 3-5 h after activation. Oocytes in Treatment 1 were microinjected with pIRES2-eGFP-Brg1 vector; oocytes in Treatment 2 were microinjected with pIRES2-eGFP vector; and oocytes in Treatment 3 were not injected with any construct. Approximately 40 pL were injected at a concentration of 1 mg/mL. Following injection, lysed embryos were discarded, and the intact embryos were placed in embryo culture medium for 6 days. At this time, embryos were stained with Hoechst 33342, and the number of nuclei for each embryo was determined. Expression of the injected construct was determined by the presence of GFP 24 h following microinjection. There was a tendency for embryos that developed from oocytes injected with the Brg1 construct to have increased cell numbers at day 6 of embryo culture, as compared with the other 2 groups (21.4 ± 15, n = 12; 17.7 ± 9.6, n = 7; 16.8 ± 5.82, n = 24 for treatments 1, 2, and 3, respectively; P = 0.3). Data were analyzed using GLM procedures in SAS; embryos with cell number e8 were analyzed. There was no difference in fragmented embryos among the 3 groups. The results come from 5 independent replicates. To the best of our knowledge, this is the first time that a chromatin remodeling protein has been overexpressed in cleavage stage porcine embryos. Further experiments using an ATPase defective subunit will be performed to further examine the role that ATP dependent chromatin remodeling processes play during porcine embryogenesis.