A source for expression profiling in single preimplantation bovine embryos.

Abstract

Our knowledge of the genes active during normal preimplantation development in cattle is limited, despite the importance for further improvement of fertility and applicability of biotechniques, like in vitro production and embryo transfer. We report on the construction of cDNA libraries as a source for expression profiling in oocytes and single preimplantation cattle embryos. cDNAs were prepared from two unfertilized oocytes, single two-cell, four-cell and eight-cell, morula, and blastocyst stage embryos, respectively. The oocytes, eight-cell, morula, and blastocyst stage embryo-derived cDNAs were ligated to a lambda-based expression vector and these have complexities of 8 x 10(5), 5 x 10(5), 1 x 10(6) and 2 x 10(6) independent clones, respectively. A total of 48 clones were picked and sequenced, 62.5% (30/48) of the sequence were homologous to known transcripts from human and mouse, 18.75% (9/48) to expressed sequence tags (ESTs) of human and mouse origin. Novel sequences were detected at a frequency of 14.58% (7/48). PCR analyses of the embryonic libraries for specific genes revealed transcripts for genes including housekeeping genes (GAPDH and beta-actin), developmental genes (OCT-4, IGF-I receptor and homeodomain sequences) and genes coding for metabolic and protective enzymes (manganese superoxide dismutase, glutamine synthetase, flavin-containing mono-oxygenase, glutamate dehydrogenase, alpha-2-macroglobulin). These cDNA libraries are a valuable resource for the isolation of clones representing genes active at these early developmental stages. The ability to construct cDNA expression libraries from only a few cells will allow gene expression analyses from embryo biopsies and embryos derived by nuclear transfer procedures.