Construction of Early Human Embryo cDNA Libraries and Screening Objective Genes.
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 lambdaZipLox phage. The plasmid libraries have complexities of 2.6x10(5), 1.7x10(5) and 2.1x10(5) clones and the phage cDNA libraries have complexities of 3.4x10(6), 3.7x10(6) and 2.3x10(6) 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.
Gou
De-Ming
DM
College of Life Sciences, Wuhan University, Wuhan 430072, China. wxli@whu.edu.cn
Huang
Jian
J
Li
Wen-Xin
WX
Mao
Xin
X
Jiang
Da-He
DH
Hua
Wen-Jun
WJ
Fan
Jun-Hua
JH