Construction and characteristics of 3-end enriched cDNA library from individual embryos of cattle.
To analyze stage-specific gene expression profiles of pre-implantation embryos and evaluate potential viability, techniques were adapted to generate 3-end enriched cDNA libraries from individual embryos of cattle based on RT-PCR methodology. The reproducibility of constructing a cDNA library was tested by five independent PCR experiments with specific primers for the presence of several rare genes such as DNMT1 (DNA methylation transferase 1), DNMT2, DNMT3A, Oct-4/3 (octmer-binding transcription factor), IFN-iota, IGF-2r (insulin like growth factor 2 receptor), and the housekeeping genes, H2A and beta-actin. Results indicated repeatability and that a proportion of expressed genes in the cDNA library from an individual embryo was not affected by limited PCR amplification. From the cDNA library, 134 clones were randomly selected for sequencing and showed that structure related elements accounted for 33.5% of transcripts and the energy- and metabolism-related genes were also an important component being 11.9% in the cDNA library. Approximately 14% of genes in the library were functionally unknown including greater than 5% of genes that were likely novel because there was no identity in Genbank. The frequency of structure-related genes such as beta-actin and ribosomal proteins in the cDNA library corresponded to other reports and suggested that the cDNA library constructed by RT-PCR might be proportional to the mRNA populations. The cDNA libraries constructed from different stage embryos will provide a powerful tool to explore novel genes relevant to embryogenesis, determine the profiling of stage-specific gene expression, and evaluate the potential viability of embryos.
Long
Jian-Er
JE
Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiaotong University, 24/1400 West Beijing Road, Shanghai 200040, PR China.
He
Li-Qiang
LQ
Cai
Xia
X
Ren
Zhao-Rui
ZR
Huang
Shu-Zhen
SZ
Zeng
Yi-Tao
YT
D000818:Animals; D001755:Blastocyst / Q000737:chemistry; D002417:Cattle / Q000196:embryology / Q000235:genetics*; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D018076:DNA, Complementary; D004622:Embryo, Mammalian / Q000737:chemistry*; D020869:Gene Expression Profiling; D015723:Gene Library; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases