Construction and characterization of a cDNA library from 4-week-old human embryo.
Development and differentiation studies of early human embryos have been severely impeded by general difficulties in obtaining suitable samples. In order to isolate and identify new genes expressed during early human development, we constructed and characterized a PCR-based cDNA library using a 4-week-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, containing beta-actin, tPA and LINE1 repetitive sequences at the expected frequencies as in other conventionally constructed and PCR-based cDNA libraries. PCR analyses of the library for specific genes have also revealed the presence of cDNAs for developmentally important genes such as CD59, MCP, Quox-1 and ZNF268. Among the 70 randomly selected cDNA clones, 53% encoded previously known genes, 26% matched with anonymous sequences, and 17% showed no sequence similarity and were designated as human early embryo-specific ESTs. These results demonstrate the sequence complexity and relatively low redundancy of our cDNA library. Furthermore, approximately 40% of those randomly 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 expression and regulatory mechanisms that underlie the early process of human embryogenesis.
Gou
D M
DM
College of Life Sciences, Wuhan University, Wuhan 430072, PR China.
Chow
L M
LM
Chen
N Q
NQ
Jiang
D H
DH
Li
W X
WX
D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D030561:Databases, Nucleic Acid; D004587:Electrophoresis, Agar Gel; D004622:Embryo, Mammalian / Q000378:metabolism*; D020224:Expressed Sequence Tags; D018507:Gene Expression Regulation, Developmental; D015723:Gene Library; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D012333:RNA, Messenger