Construction of a Minicircle DNA Vector In Order Applicable for Generation of Induced Pluripotent Stem Cells

• Published in: Cell journal (Yakhteh) Vol. 13; p. 43
• Main Authors: Sanei, N, Ghaedi, K, Khazaie, Y, Dormiani, K, ouzanfar, M, Rezaie, N, Tavassoli, M, Esfahani, NMH
• Format: Journal Article
• Language: English
• Published: 01.01.2011
• Subjects: Arabinose; Cell culture; Data processing; DNA; Expression vectors; genomics; Green fluorescent protein; Head; Integrase; Integration; Plasmids; Primers; Stem cells; Tails; Transgenes
• ISSN: 2228-5806

Objective: Minicircle vectors are DNA molecules that lack bacterial backbone so composed of a eukaryotic expression cassette. Minicircle vectors are capable of higher and longer expression compared to plasmids. Minicircle DNA vectors show rare integration of transgene into genomic DNA. Therefore DNA minicircle will be elutable in cells, thus Minicircle vectors are ideal vectors for generating iPS cells. Materials and Methods: At the first step, egfp (enhanced green fluorescent protein) CDS was amplified using the plasmid pEGFP-Cl as a template and primers introducing integrase recognition sequences at both of head and tail of amplified fragment. Amplified fragment was then inserted into Snal site of pBAD.gHIA. Recombinant plasmid pBAD. EGFP was amplified by transforming into Ecoli cells. At the next step, Phi C31 integrase cDNA was amplified using plasmid pCMV-INT as template and appropriate primers introducing BamHI and SacI sites at head and tail respectively. This amplified fragment was then inserted into pBAD. EGFP, resulting in formation of a new vector termed pBAD. Phi C31. EGFP to generate minicircle DNA. An induction by arabinose in the culture of pBAD. Phi C31. EGFP transfomrmed Ecoli cells, caused generation of minicircle DNA carrying egfp in to the eukaryotic cells. Results: New recombinant plasmid pBAD. Phi C31. EGFP was constructed successfully and its potency for generation of minicircle DNA in Ecoli cells was verified after induction by arabinose to stimulate the expression of integrase gene. Conclusion: As minicircle DNA rarely integrate to the host cells, are considered as an ideal system to express the interest genes in the target cells. Recent reports have indicated the application of minicircle DNA for generation of iPS cells. In order to set the same approach for generating iPs, the present study carried out to produce a mother plasmid carrying both of integrase coding gene and a gene marker our data have indicated that such plasmid was successfully constructed and generated a minicicle DNA. This plasmid would be applicable for further studies which is ongoing.