[Construction of fetal mesenchymal stem cell cDNA subtractive library].
To identify differentially expressed genes between fetal mesenchymal stem cell (MSC) and adult MSC, especially specified genes expressed in fetal MSC, a cDNA subtractive library of fetal MSC was constructed using suppression subtractive hybridization (SSH) technique. At first, total RNA was isolated from fetal and adult MSC. Using SMART PCR synthesis method, single-strand and double-strand cDNAs were synthesized. After Rsa I digestion, fetal MSC cDNAs were divided into two groups and ligated to adaptor 1 and adaptor 2 respectively. Results showed that the amplified library contains 890 clones. Analysis of 890 clones with PCR demonstrated that 768 clones were positive. The positive rate is 86.3%. The size of inserted fragments in these positive clones was between 0.2 - 1 kb, with an average of 400 - 600 bp.


CONCLUSIONS
SSH is a convenient and effective method for screening differentially expressed genes. The constructed cDNA subtractive library of fetal MSC cDNA lays solid foundation for screening and cloning new and specific function related genes of fetal MSC.
Yang
Li
L
Department of Stem Cell, Beijing Institute of Transfusion Medicine, Beijing 100850, China.
Wang
Dong-Mei
DM
Li
Liang
L
Bai
Ci-Xian
CX
Cao
Hua
H
Li
Ting-Yu
TY
Pei
Xue-Tao
XT
D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005333:Fetus; D015723:Gene Library; D006801:Humans; D008648:Mesoderm / Q000166:cytology / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*
D018076:DNA, Complementary; D015252:Deoxyribonucleases, Type II Site-Specific; C040158:GTAC-specific type II deoxyribonucleases