[Screening of a hepatic stellate cells subtracted cDNA library of differentially expressed genes in mice with schistosomiasis japonica].
OBJECTIVE
To construct and screen a hepatic stellate cells(HSCs) subtracted cDNA library, in order to seek differentially expressed genes in mice infected with Schistosoma japonicum.


METHODS
HSCs were isolated from mouse as targets, and cDNA fragments of normal mouse were compared to those of S. japonicum infected mice to find dif ferentially expressed genes by technique of suppression subtractive hybridization(SSH). Differentially expressed cDNA fragments were then directly inserted into T/A cloning vector to set up the subtractive library. Amplification of the library was carried out with transformation of DH5alpha. A subtracted cDNA library was constructed and then hybridized with forward and backward subtracted probes for differential screening. One hundred positive bacterial clones were randomly selected for se quenching and BLAST analysis. Finally, virtual Northern blot confirmed such differential expression.


RESULTS
The amplified library contained more than 400 positive bacterial clones. Random analysis of 100 clones with restriction enzyme digestion showed that all clones contained 200-600 bp inserts. 76 ESTs were obtained with 70 related to fibrosis caused by schistosomiasis or other etiological factors. Other 6 ESTs were not found in PubMed.


CONCLUSIONS
The subtracted cDNA library of differentially expressed genes of HSC in normal mice and schistosome-infected mice was constructed successfully with SSH and T/A cloning techniques.
Zheng
Min
M
Institute of Infectious Diseases, First Affiliated Hospital, Medical School, Zhejiang University, Hangzhou 310003, China.
Chen
Feng
F
Cai
Wei-min
WM
Liu
Rong-hua
RH
D000818:Animals; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D055166:Hepatic Stellate Cells / Q000166:cytology / Q000378:metabolism* / Q000469:parasitology; D008103:Liver Cirrhosis / Q000235:genetics / Q000469:parasitology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012554:Schistosomiasis japonica / Q000235:genetics* / Q000469:parasitology; D017422:Sequence Analysis, DNA
D004247:DNA