Isolation of nascent messenger RNA from mouse preimplantation embryos
Isolation of nascent messenger RNA from mouse preimplantation embryos
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DOI：
10.1095/biolreprod.104.031906
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发表时间：
2004-12-01
影响因子：
3.6
通讯作者：
Aoki, F
中科院分区：
文献类型：
--
作者：
Kageyama, SI;Nagata, M;Aoki, F
合子基因组在小鼠胚胎中的表达始于单细胞晚期，其调控动态变化到双细胞期的晚期。为了理解这一过程，重要的是积累在每个发育阶段的任何给定时刻转录的基因的图谱。然而，由于大量母体mRNA在胚胎中积累以维持早期发育，因此很难确定基因激活后新合成的mRNA的分布情况。为了克服这一困难，我们建立了一种新的方法，从大量先前存在的mRNA库中分离出新生mRNAs。简而言之，程序如下。胚胎电渗透并负载5-溴-5‘-三磷酸(BrUTP)。用识别Bru的抗体进行免疫沉淀法，获得了掺入Bru的新生mRNA。从分离的mRNA中合成了c DNA，并用实时荧光定量聚合酶链式反应检测了其丰度。利用这种方法，我们检测了两细胞小鼠胚胎中新合成的ELF-1A、MuERV-L和Cyclin-A2转录本的数量，并将它们与总mRNA库中的这些转录本的数量进行了比较。随着时间的推移，每个转录本在新生mRNA部分和总mRNA池中的数量变化不同，表明这种方法可以用来获得在发育过程中转录的基因的图谱。
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 realtime PCR. Using this method, we examined the amounts of newly synthesized elf-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.