Embryo

Isolation of Nascent Messenger RNA from Mouse Preimplantation Embryos

Abstract
The expression of the zygotic genome starts at the late one-cell stage in mouse embryos, and its regulation changes dynamically until the late two-cell stage. To understand this process, it is important to accumulate the profiles of the genes transcribed at any given instant of each stage of development. However, because large amounts of maternal mRNA accumulate in embryos to sustain early development, it is difficult to determine the profile of newly synthesized mRNA just after gene activation. To overcome this difficulty, we established a novel method of isolating nascent mRNA from the large pool of pre-existing mRNA. Briefly, the procedure was as follows. Embryos were electrically permeabilized and loaded with 5-bromouridine-5'-triphosphate (BrUTP). Nascent mRNA with incorporated BrU was isolated by immunoprecipitation with an antibody recognizing BrU. cDNA was synthesized from the isolated mRNA, and its abundance was evaluated using semi-quantitative real-time PCR. Using this method, we examined the amounts of newly synthesized eIF-1A, MuERV-L, and cyclin A2 transcripts in two-cell mouse embryos and compared them with the quantities of these transcripts present in the total mRNA pool. The amount of each transcript in the nascent mRNA fraction and in the total mRNA pool changed differently over time, demonstrating that this method can be used to obtain profiles of genes transcribed during development.

Key words: Embryo • Early development • Fertilization • Gene regulation

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