A RAPID METHOD FOR MESSENGER-RNA DETECTION IN SINGLE-CELL BIOPSIES FROM PREIMPLANTATION-STAGE BOVINE EMBRYOS

Major questions concerning the control of development and gene express ion at the cellular level are still unanswered. Nowhere is this more e vident than during the earliest stages of development and embryogenesi s. This study describes the detection of specific gene transcripts in single cells derived from bovine embryos. Following in vitro fertiliza tion (IVF) and in vitro culture (IVC) of bovine embryos, small groups of cells and even single blastomeres from 32 to 64-cell embryos were m icromanipulated into individual tubes for analysis of cytoplasmic RNAs . Reverse transcriptase-PCR was applied to cell lysates for the amplif ication of beta-actin mRNA transcripts. Primers were designed to flank an intron expected to be present within genomic DNA sequences, thus a llowing for simple differentiation between DNA- and RNA-derived amplif ication products. Using a 50-cycle amplification profile, a 260 bp ban d could. be seen as a PCR product derived from a single blastomere fol lowing electrophoresis in an ethidium bromide-stained agarose gel. The identity of the band was verified by DNA sequence determination and d iagnostic restriction digestion. Lysates derived from single blastomer es in this way have been used for simultaneously phenotyping multiple RNA products. This capability allows the spatial analysis of gene expr ession and development within embryos from the earliest stages of cell ular differentiation.