Construction and characterization of a cDNA library from human embryonic stem cells

[Objective] To construct a cDNA library of human embryonic stem(hES) cell for screening,cloning and studying new genes.[Methods] Thomson-derived human embryonic stem cells were cultured and isolated.Total RNA was extracted from hES cell and mRNA was purified using mRNA oligo(dT) magnetic particle purification technique.The first and second strand cDNA was synthesized by reverse transcriptase and DNA polymerase I respectively.The ds-cDNA termini was blunted with pfu DNA polymerase.The blunted cDNAs were added EcoRⅠadaptor and then digested by XhoⅠ.The cDNA fragments longer than 0.5 kb were collected by Sepharose CL2B chromatography and ligated to Uni-ZAP XR expression vector.The ligated products were packaged in vitro and the lambda phage particles infected the host strains XL1-BlueMRF' to produce an unamplified library.The library was amplified and tittered.The size of cDNA fragment was evaluated by EcoRⅠand XhoⅠ restriction endonuclease digestion.[Results] The efficiency of the primary library was 1.2 ×106 recombinant and the amplified library was 1.0 ×109 pfu /mL.The percentage of positive recombinant clones was 98.5%.The length of the inserted cDNA fragment was over 1 kb.[Conclusion] The quality of the constructed hES cDNA library is excellent and helpful to screen new genes.