Construction and characterization of a cDNA library from 4-week-old human embryo

Development and differentiation studies of early human embryos have been se verely impeded by general difficulties in obtaining suitable samples. In or der to isolate and identify new genes expressed during early human developm ent, we constructed and characterized a PCR-based cDNA library using a 4-we ek-old chorion-free human embryo. The constructed cDNA library contained 6. 3 x 10(6) directional recombinants, and its insert size ranged from 0.4 to 1.8 kb. The cDNA library proportionally represents the mRNA population, con taining beta -actin, tPA and LINE1 repetitive sequences at the expected fre quencies as in other conventionally constructed and PCR-based cDNA librarie s. PCR analyses of the library for specific genes have also revealed the pr esence of cDNAs for developmentally important genes such as CD59, MCP, Quox -1 and ZNF268. Among the 70 randomly selected cDNA clones, 53% encoded prev iously known genes, 26% matched with anonymous sequences, and 17% showed no sequence similarity and were designated as human early embryo-specific EST s. These results demonstrate the sequence complexity and relatively low red undancy of our cDNA library. Furthermore, approximately 40% of those random ly analyzed clones contained full-length encoding regions. To our knowledge , this is the first description of the PCR-based cDNA library from a 4-week -old chorion-free human embryo, and the presence of novel sequences within this library makes it a valuable and unique resource for studying gene expr ession and regulatory mechanisms that underlie the early process of human e mbryogenesis. (C) 2001 Elsevier Science B.V. All rights reserved.