Isolation of nascent messenger RNA from mouse preimplantation embryos.
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 at 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 preexisting 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. The cDNA was synthesized from the isolated mRNA, and its abundance was evaluated using semiquantitative 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.
Kageyama
Shun-Ichiro
S
Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan.
Nagata
Masao
M
Aoki
Fugaku
F
D000818:Animals; D001755:Blastocyst / Q000378:metabolism*; D002478:Cells, Cultured; D003199:Computer Systems; D019925:Cyclin A / Q000235:genetics; D018076:DNA, Complementary / Q000378:metabolism; D046149:Embryo Culture Techniques; D015977:Eukaryotic Initiation Factor-1 / Q000235:genetics; D005821:Genetic Techniques / Q000592:standards; D047468:Immunoprecipitation; D051379:Mice; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014544:Uridine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism
D019925:Cyclin A; D018076:DNA, Complementary; D015977:Eukaryotic Initiation Factor-1; C499132:MuERV-L protein, mouse; D011506:Proteins; D012333:RNA, Messenger; C026022:eukaryotic peptide initiation factor-1A; C018009:5-bromouridine triphosphate; D014544:Uridine Triphosphate