| 研究概要 |
We have isolated four recombinant phage from a TT2 embryonic stem cell genomic library(Kindly provided by Dr.T.Yagi,Okazaki Kokuritsu Kyodo Kenkyu Kikan,Seirigaku Kenkyujo)that contain sequences that span the Zif268gene. We are currently using DNA segments derived from one of these phage to construct a targeting vector that will allow the endogenous Zif268 genes in TT2 embryonic stem cells to be replaced by the E.coli bet-galactosidase gene.This targeting vector will contain DNA segments derived from the 5′and3′ sides of the Zif268 gene flanking a beta-galactosidase+neomycin resistance gene cassette,which was constructed in Dr.Obata′s laboratory.The targeting vector will also contain a diphtheria toxin gene located downstream from the Zif2683′ sequences,which will aid in the selection of TT2 cells that have acquired the neomycin resistance gene by homologous recombination.
We have constructed a Zif268/green fluorescent protein expression vector for studies of inducible gene expression in living PC12D cells.This expression vector encodes a fusion protein that contains the first 13 amino acids of Zif268 followed by a 4 amino acid bridge and the entire green fluorescent protein.The green fluorescent protein contains the serine 65 to threonine(S65T)point mutation,which has been reported to greatly enhance the fluorescence and rate of folding of this protein.Expression of the Zif268/GFP fusion protein has been placed under the control of a 470 base pain segment of the of the Zif268 promoter.
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