The central thrust of our laboratory is placed upon the production and analysis of transgenically modified animals for production of therapeutic proteins. The key areas include,
In the Past...
Transgenesis: Classical approaches for producing transgenic animals require labor-intensive, time-consuming, and expensive methods but have low transgenic efficiency and high mosaicism rate. In the past, we developed a simplified method for producing transgenic (EGFP gene) porcine embryos by microinjecting DNA construct into unfertilized metaphase oocytes that are subsequently fertilized in vitro (termed oocyte mediated gene transfer or OMGT). In addition, we have also used retroviral vectors (EGFP gene) and conventional plasmid vectors (GCSF gene) for production of transgenic porcine and bovine cloned embryos by intraspecies/interspecies/intrebreed somatic cell nuclear transfer.
Nuclear Reprogramming: We have demonstrated that the imprinting mechanism of IGF2 and H19 genes in porcine is similar to those of other species and cloned embryos and offspring show aberrant DNA methylation pattern at DMRs of IGF2 gene loci. Acetylation at Histone 3 Lysine 9 residue has also been shown to be a key player in epigenetic modification that display marked variability during embryonic preimplantation development. Dynamic changes and interplay between these two epigenetic mechanisms could be crucial for embryonic development during the early preimplantation period.
Assisted Reproductive Technologies: We have found that lipid peroxidation is a key culprit of blastomeric fragmentation of porcine embryos. A more in-depth analysis revealed that counting the number of blastomeres on Day 2 of in vitro culture could be used as a valuable non-invasive tool for morphological selection of good quality embryos for transfer. Abnormal cell division was positively correlated with chromosomal aberration, as revealed by fluorescent in situ hybridization technique.
Embryo Proteomics: We had utilized proteomic approach to understand the mechanism underlying embryonic development and identification of biomarkers of embryo quality. Utilizing the LC-MS/MS technology, we are the first to globally sequence and quantify more than 3000 protein in porcine zygotes. Among them several are potential candidates of marker for embryonic development and are under study.