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OJVRTM

Online Journal of Veterinary Research

 

Volume 25 (3): 207-213, 2021.


Lentiviral gene vector transformation in Escherichia coli derived H5α, JM109 and Stbl4 cells

 

Azam Mokhtari1, 2, Shekoofeh Rezayi1

 

1Department of Pathobiology, Faculty of Veterinary Medicine, 2Research Institute of Zoonotic Diseases, Shahrekord University, Shahrekord, Iran

 

ABSTRACT

 

Mokhtari A, Rezayi S., Lentiviral gene vector transformation in Escherichia coliderived H5α, JM109 and Stbl4 cells, Onl J Vet Res., 25 (3):207-213, 2021. Bacterial cell transformation is essential for gene cloning. Although plasmid gene transfer recombinant lentivectors are highly effective, they do not transform readily in most cells. E coli derived Stbl2 and Stbl3 strains maintain structure of lentiviral plasmid pRRL.SIN.cPPT.PGK/Oligo2-IRES-DsRed-WPRE and boost colony growth. However, Stbl4 appears to be even more efficient to justify further study. We thus investigated plasmid transformation in DH5α, JM109 and Stbl4 cells derived from Escherichia coli. To transform recombinant plasmids in DH5α, Stbl4 and JM109 and confirm the process we used negative controls without plasmid and a positive control by heat shock method with Amp resistance gene. We calculated transformation cloning units by bacterial suspension culture under identical cell conditions and ran 20 replications for each cell. Calcium chloride precipitation and thermal shock was done to determine transform efficiency by colonies yielding 0.26 106 for DH5α, 0.89 106 for JM109 and 1.57 106 for Stbl4 cfu / g. Our results suggest that optimal cell for pWPI lentiviral vector was DH5α and Stbl4 most suitable for recombinant pCDH vectors.

 

Keywords: Transformation, competent cell, DH5α, JM109, Stbl4, colony formation unit.


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