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Construction of the mammalian expressing vector pEGFP-N1-P53 and its expression successful in chicken fibroblast cells and blastoderm.

Abstract
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.
AuthorsZ Song, Z H Li, X Q Lei, T S Xu, X H Zhang, Y J Li, G M Zhang, S M Xi, Y B Yang, Z G Wei
JournalGenetics and molecular research : GMR (Genet Mol Res) Vol. 14 Issue 1 Pg. 931-9 (Feb 02 2015) ISSN: 1676-5680 [Electronic] Brazil
PMID25730031 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Recombinant Fusion Proteins
  • TP53 protein, human
  • Tumor Suppressor Protein p53
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
Topics
  • Animals
  • Blastoderm (growth & development, metabolism)
  • Chick Embryo
  • Fibroblasts (metabolism)
  • Gene Expression Regulation, Developmental
  • Genetic Vectors
  • Green Fluorescent Proteins (genetics)
  • Humans
  • Recombinant Fusion Proteins (biosynthesis, genetics)
  • Tumor Suppressor Protein p53 (biosynthesis, genetics)

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