Establishment of a targeting protein for Xenopus kinesin-like protein 2 C' terminal SBP-3 x Flag tagged HCT 116 colorectal cancer cell model.
- Author:
Ze-Bin HUANG
1
;
Ze-Yan ZHANG
;
Xiao-Dong ZHANG
;
Shi-Ying MIAO
;
Lin-Fang WANG
;
Run-Lei DU
Author Information
- Publication Type:Journal Article
- MeSH: Cell Cycle Proteins; genetics; Colorectal Neoplasms; genetics; pathology; Dependovirus; genetics; Gene Targeting; Genetic Vectors; HCT116 Cells; Humans; Microtubule-Associated Proteins; genetics; Nuclear Proteins; genetics
- From: Acta Academiae Medicinae Sinicae 2011;33(6):624-628
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo develop a targeting protein for Xenopus kinesin-like protein 2 (TPX2) C' terminal SBP-3 x Flag-tagged HCT 116 cell model.
METHODSHomologous arms were amplified by polymerase chain reaction (PCR), and then the adeno-associated virus (AAV) -targeting vector of TPX2 was constructed. HCT 116 cells were targeted after the viruses were packaged. Positive cell clones with neomycin resistance gene were obtained by G418 and PCR screening. Finally, the neomycin gene cassette was excised after the targeted clones were infected with adenovirus expressing Cre-recombinase, and the TPX2 C' terminal SBP and 3 x Flag endogenous double-tagged HCT 116 cells were obtained by PCR screening.
RESULTSTwo positive cell clones with neomycin resistance gene were obtained by PCR screening. The positive clones with neomycin resistance gene excised were obtained by Cre adenovirus infection, and the knock-in of SBP-3 x Flag gene was verified by Western blot analysis.
CONCLUSIONThe TPX2 C' terminal SBP-3 x Flag tagged HCT 116 cell model was successfully established.
