Construction of a new minicircle DNA carrying an enhanced green florescent protein reporter gene for efficient expression into mammalian cell lines.
The presence of a bacterial backbone in conventional eukaryotic expression plasmids may cause undesirable effects by triggering the immune responses in mammals and repression of episomal transgene expression. To avoid these problems, researchers have proposed the use of minicircle DNAs which are episomal vectors that have lost their bacterial backbone using a site-specific recombinase mediated recombination. In the present study, we have constructed a new minicircle DNA vector that carries an enhanced green florescent protein (EGFP) reporter gene using phage ΦC31 integrase-mediated recombination and homing endonuclease ISceI-mediated purification in E. coli. ΦC31 integrase expression was under the control of the araBAD promoter, whereas ISceI endonuclease was controlled by the tac promoter. This vector was transfected into CHO-K1 cells, which showed transient expression of EGFP up to 14 generations. Similar results were obtained upon transient transfection into HEK cells. In addition, PCR results on genomic DNA, demonstrated the EGFP-minicircle was episomal and did not integrate into the host genome. Our constructed parental plasmid expresses EGFP and could be used for the generation of episomal minicircle DNA with intent to carry out transient transfection of interested DNA fragments into the eukaryotic cells for various purposes.
Department of Biology, School of Sciences, University of Isfahan, Isfahān, Iran, nafiseh.sanei@gmail.com.
Nafiseh
N
Sanei Ata-Abadi
Kianoush
K
Dormiani
Yahya
Y
Khazaie
Kamran
K
Ghaedi
Mahboobeh
M
Forouzanfar
Liana
L
Lachinani
Naeimeh
N
Rezaei
Abbas
A
Kiani-Esfahani
Mohammad Hossein
MH
Nasr-Esfahani
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D003412:Cricetulus; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism; D004270:DNA, Circular / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic
D004270:DNA, Circular; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C077570:Site-specific recombinase; D004262:DNA Restriction Enzymes