PARENTAL IMPRINTING STUDIED BY ALLELE-SPECIFIC PRIMER EXTENSION AFTER PCR - PATERNAL-X CHROMOSOME-LINKED GENES ARE TRANSCRIBED PRIOR TO PREFERENTIAL PATERNAL X-CHROMOSOME INACTIVATION
PARENTAL IMPRINTING STUDIED BY ALLELE-SPECIFIC PRIMER EXTENSION AFTER PCR - PATERNAL-X CHROMOSOME-LINKED GENES ARE TRANSCRIBED PRIOR TO PREFERENTIAL PATERNAL X-CHROMOSOME INACTIVATION
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DOI：
10.1073/pnas.89.21.10469
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发表时间：
1992-11-01
影响因子：
11.1
通讯作者：
RIGGS, AD
中科院分区：
文献类型：
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作者：
SINGERSAM, J;CHAPMAN, V;RIGGS, AD
在早期小鼠发育过程中，胚胎外细胞中父方X染色体的优先失活是亲本印记的一个例子，但尚未在转录水平上进行研究，因为测量RNA水平的标准方法不允许在单个早期胚胎中检测等位基因特异性RNA。我们试图确定父方等位基因的X染色体连锁基因3-磷酸甘油酸激酶1（Pgk-1），这是非常接近的X染色体失活的中心，是转录前分化的胚外谱系。以前的报道表明，在杂合子胚胎中，由父方X染色体编码的磷酸甘油酸激酶1等位酶的出现延迟，直到相应的母方等位酶出现后2天。我们报告通过使用逆转录/PCR为基础的方法，允许等位基因特异性RNA的定量测量所获得的结果。该测定法是足够灵敏的定量分析，在单个胚胎的等位基因特异性转录本不同，只有一个核苷酸。我们已经使用这种方法来分析在Pgk-1和Hprt [次黄嘌呤（鸟嘌呤）磷酸核糖转移酶]基因座杂合的小鼠胚胎，我们发现，个别8细胞和囊胚胚胎表达Hprt和Pgk-1父系转录本，因为合并的2- 4细胞胚胎。鉴于Pgk-1基因在酶水平上的父系表达的明显时间延迟，这些结果进行了讨论。
The preferential inactivation of the paternal X chromosome in extraembryonic cells during early mouse development is an example of parental imprinting, but it has not been studied at the transcriptional level because standard methods of measuring RNA levels do not allow detection of allele-specific RNAs in individual early embryos. We sought to determine whether the paternal allele of the X chromosome-linked gene for 3-phosphoglycerate kinase 1 (Pgk-1), which is located very near the center of X chromosome inactivation, is transcribed prior to differentiation of extraembryonic lineages. Previous reports indicated that in heterozygous embryos there is a delay in the appearance of the phosphoglycerate kinase 1 allozyme encoded by the paternal X chromosome until 2 days after the appearance of the corresponding maternal allozyme. We report results obtained by use of a reverse transcription/PCR-based method which allows the quantitative measurement of allele-specific RNA. The assay is sensitive enough for the quantitative analysis in single embryos of allele-specific transcripts differing by only one nucleotide. We have used this assay to analyze mouse embryos heterozygous at the Pgk-1 and Hprt [hypoxanthine (guanine) phosphoribosyltransferase] loci, and we find that individual 8-cell and blastocyst embryos express both Hprt and Pgk-1 paternal transcripts, as do pooled 2- to 4-cell embryos. These results are discussed in view of the apparent temporal delay in paternal expression of the Pgk-1 gene at the enzyme level.