Construction of Early
Human Embryo cDNA Libraries and Screening Objective Genes
GOU De-Ming, HUANG Jian, LI Wen-Xin, MAO Xin and JIANG Da-He
( College of Life Sciences, Wuhan University, Wuhan 430072, China
)
HUA Wen-Jun and FAN Jun-Hua
( Biotechnology Research Center, Hubei Academy of Agricultural Sciences,
Wuan 430064, China )
Abstract The
construction, evaluation, and application of cDNA libraries from 3-, 4-, and
5-week-old human embryos are described. Total RNAs were extracted from whole
embryos using a modified single-step method. mRNA purified by two passes
through oligo(dT) columns was reverse-transcripted into single-stranded cDNA.
Alkaline agarose electrophoresis showed that the double-strand cDNA fragments
ranged from 0.4－9.0 kb and most of them were in the range
of 1.0－2.0
kb. After separation on SizeSep 400 Spun columns to eliminate excess adaptors
and small cDNA fragments(less than 400 bp), the cDNAs were ligated into pSPORT1
plasmid and λZipLox phage. The plasmid libraries have complexities of 2.6×105,
1.7×105 and 2.1×105 clones； and the phage cDNA libraries
have complexities of 3.4×106, 3.7×106 and 2.3×106
clones, respectively. Three whole length cDNAs encoding human CD59, MCP and DAF
were amplified by PCR using 3-week-old phage library as templates, and human tPA
gene with whole length cDNA was screened from 4-week-old plasmid library by
hybridization. It was shown that these libraries are of high quality and are
suitable to screen rarely expressed genes. The libraries are a valuable source
for the study of novel gene expression during human development.
Key Words early human embryo； cDNA library； gene clone
Corresponding author： Tel, 86-27-87684380； Fax, 86-27-87882661； e-mail, [email protected]