Developmental expression of specific genes detected in high-quality cDNA libraries from single human preimplantation embryos
Developmental expression of specific genes detected in high-quality cDNA libraries from single human preimplantation embryos
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DOI：
10.1016/s0378-1119(99)00329-7
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发表时间：
1999-09-17
期刊：
影响因子：
3.5
通讯作者：
Monk, M
中科院分区：
文献类型：
--
作者：
Adjaye, J;Bolton, V;Monk, M
我们描述了一种改进的高灵敏度方法，用于生成 cDNA 文库，其中含有高比例的 cDNA，富含来自单个人类植入前胚胎和 10 周龄全胎儿的 5' 编码序列。使用连接至磁珠的寡聚 (dT) 分离胚胎 mRNA。第一链 cDNA 合成直接在结合的 mRNA 上进行，然后进行 PCR 设计以扩增整个合成的 cDNA 分子。文库的复杂性在 10(5) 和 10(6) 个独立克隆之间。 cDNA的平均大小为1.0 kb，大小范围为0.5-3.0 kb。对胚胎文库中特定基因的 PCR 分析揭示了已知在植入前阶段转录的基因的转录本，例如印记基因 SNRPN、发育基因 WNT11、HOX、OCT-1 和胚胎 OCT-4、细胞骨架基因 keratin-18 和 β-肌动蛋白、细胞周期基因 C-MOS 以及管家基因 GAPDH 和 HPRT。随机克隆的测序显示存在多种序列，例如人绒毛膜促性腺激素、泛素、TFIIA、鸟嘌呤核苷酸结合蛋白（β亚基）、膜联蛋白 I（编码驱动蛋白样蛋白的基因）和 TWIST（编码与 Saethre-Chotzen 综合征（以颅面和肢体为特征）有关的基本螺旋-环-螺旋 (bHLH) 转录因子）异常）。这些随机分析的克隆中大约有 40% 是全长的。除了与 GenBank 和 dbEST 数据库中已知 EST（表达序列标签）相匹配的 cDNA 外，在随机挑选的克隆中，还以 16% 的频率检测到新序列。这些文库是宝贵的资源，提供代表人类植入前发育过程中表达的基因的较长 cDNA。 (C) 1999 Elsevier Science B.V. 保留所有权利。
We describe an improved highly sensitive method for generating cDNA libraries containing a high proportion of cDNAs enriched with 5'-coding sequences from single human preimplantation embryos and a 10 week old whole foetus. The embryonic mRNA was isolated using oligo-(dT) linked to magnetic beads. First-strand cDNA synthesis was carried out directly on the bound mRNA, followed by PCR designed to amplify the cDNA molecules synthesized in their entirety. The complexities of the libraries are between 10(5) and 10(6) independent clones. The average cDNA size is 1.0 kb, and the size range is 0.5-3.0 kb. PCR analysis of the embryonic libraries for specific genes has revealed transcripts for genes known to be transcribed in preimplantation stages, such as the imprinted gene SNRPN, developmental genes WNT11, HOX, OCT-1 and the embryonic OCT-4, cytoskeletal genes keratin-18 and beta-actin, the cell cycle gene C-MOS, and housekeeping genes GAPDH and HPRT. Sequencing of random clones showed the presence of a variety of sequences, such as human chorionic gonadotrophin, ubiquitin, TFIIA, guanine nucleotide-binding protein (beta-subunit), annexin I, a gene encoding a kinesin-like protein, and TWIST, which encodes a basic helix-loop-helix (bHLH) transcription factor implicated in Saethre-Chotzen syndrome (characterized by craniofacial and limb anomalies). Approximately 40% of these randomly analysed clones were full length. In addition to cDNAs matching known ESTs (Expressed Sequence Tags) in the GenBank and dbEST databases, novel sequences were detected at a frequency of 16% of randomly picked clones. The libraries are a valuable resource, providing longer cDNAs representing genes expressed during human preimplantation development. (C) 1999 Elsevier Science B.V. All rights reserved.