[Expression vector for the inhibitor of growth-1 gene is constructed and the NLS-GFP fusion protein expresses in MRC-5 cells].
OBJECTIVE
To construct the NLS(ING1)-GFP vector, transfer it into MRC-5 cells and establish a cell model expressing NLS (ING1)-GFP fusion protein.


METHODS
Firstly, cDNA fragment of nuclear locating sequence (NLS) of inhibitor of growth-1 gene (ING1) was gained by RT-PCR and inserted into multi-clone site of pEGFP-C1 to construct the NLS (ING1)-GFP expression vector. Then the vector was used to transfect the MRC-5 cells to observe the subcellular signal localization of green fluorescence protein (GFP).


RESULTS
We successfully constructed the expressing vector of NLS (ING1)-GFP fusion protein. After transferring the fusion expressing vector into MRC-5 cells, we observed that green fluorescence signal located in the cell nucleus. However, the green fluorescence signal located in the cytoplasm in MRC-5 cells transfected with pEGFP-C1 control only expressing GFP.


CONCLUSIONS
In living cells, physiologically p33 ING1b locates absolutely in nucleus. The p33(ING1b) NLS plays a decisive role in the transporting process of subcellular localization.
Sun
Zhao-ming
ZM
Department of Neuropathology, Neurology Institute, Tianjin Medical University General Hospital, Tianjin, 300052 PR China.
Yu
Shi-zhu
SZ
Zhang
Wen-zhi
WZ
Kang
Chun-sheng
CS
Zhou
Hong-xu
HX
Huang
Hui-ling
HL
An
Tong-ling
TL
D001483:Base Sequence; D002460:Cell Line; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D000076023:Inhibitor of Growth Protein 1; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
C501503:ING1 protein, human; D000076023:Inhibitor of Growth Protein 1; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D049452:Green Fluorescent Proteins