Construction of the mammalian expressing vector pEGFP-N1-P53 and its expression successful in chicken fibroblast cells and blastoderm.
The enhanced green fluorescent protein (EGFP) pEGFP-N1-P53 eukaryotic expression vector, which contains the human tumor suppressor p53, was constructed and transfected into chicken fibroblast cells and stage-X blastoderm to analyze the transfection efficiency. The complementary DNA of the human p53 gene was cloned by reverse transcription-polymerase chain reaction from human peripheral blood and inserted into the pEGFP-N1 vector by HindIII and BamHI double digestion. The pEGFP-N1-P53 vector was transfected into chicken embryo fibroblasts by Lipofectamine 2000 liposomes, and the transfection efficiency was analyzed by fluorescence microscope after 36 h of transfection. The stage-X blastoderm was also transfected by blastoderm injection using Lipofectamine 2000 liposomes at room temperature after 12-24 h; then hatching occurred until seventh day, and the transfection efficiency was analyzed by fluorescence microscope in the dead embryo. A total of 90 hatching eggs were transfected by the pEGFP-N1-P53 vector, and 20 chicken embryos expressed the reporter gene, which indicated that recombinant pEGFP-N1-P53 could be transfected and expressed in stage-X blastoderm by liposomes. Chicken embryo fibroblasts were transfected and expressed the reporter gene. The pEGFP-N1-P53 vector was constructed successfully and could be transfected and expressed in chicken embryo fibroblasts and stage-X blastoderms efficiently.
College of Animal Science, Henan University of Science and Technology, Luoyang, Henan, China.
Z
Z
Song
College of Animal Science, Henan University of Science and Technology, Luoyang, Henan, China.
Z H
ZH
Li
College of Animal Science, Henan University of Science and Technology, Luoyang, Henan, China xueqinlei@163.com.
X Q
XQ
Lei
College of Animal Science, Henan University of Science and Technology, Luoyang, Henan, China.
T S
TS
Xu
College of Animal Science, Henan University of Science and Technology, Luoyang, Henan, China.
X H
XH
Zhang
College of Animal Science, Henan University of Science and Technology, Luoyang, Henan, China.
Y J
YJ
Li
The First Affiliated Hospital of Henan University of Science and Technology, Luoyang, Henan, China.
G M
GM
Zhang
Institute of Medicine, Henan University of Science and Technology, Luoyang, Henan, China.
S M
SM
Xi
College of Animal Science, Henan University of Science and Technology, Luoyang, Henan, China.
Y B
YB
Yang
College of Animal Science, Henan University of Science and Technology, Luoyang, Henan, China.
Z G
ZG
Wei
D000818:Animals; D001756:Blastoderm / Q000254:growth & development / Q000378:metabolism; D002642:Chick Embryo; D005347:Fibroblasts / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics*
D011993:Recombinant Fusion Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins