Open AccessFunctional efficiency of PCR vectors in vitro and at the organism level
Author(s) -
Dina Safina,
Polina I. Selina,
Marina P. Roschina,
Maria A. Karaseva,
Alexey A. Komissarov,
Ilya V. Demidyuk,
Е. Д. Свердлов,
Sergey V. Kostrov
Publication year - 2020
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0232045
Subject(s) - luciferase , plasmid , microbiology and biotechnology , biology , transfection , in vitro , reporter gene , expression vector , gene , green fluorescent protein , gene expression , recombinant dna , genetics
The functional efficiency of the expression cassettes integrated into a plasmid and a PCR- amplified fragment was comparatively analyzed after transient transfection in vitro or introduction into the developing embryo of Danio rerio . The cassettes contained the reporter genes, luciferase of Photinus pyralis ( luc ) or enhanced green fluorescent protein, under the control of the promoter of human cytomegalovirus immediate-early genes. In the in vitro system, the efficiency of the circular plasmid was 2.5 times higher than that of the PCR- amplified fragment. The effect of mutations in the expression cassette on the efficiency of the transgene expression in the PCR- amplified fragment was quantitatively evaluated. The mutations generated after 25 amplification cycles with Taq DNA polymerase decreased luciferase activity in transfected cells by 65–85%. Thus, mutations are the key factor of decreased functional efficiency of the PCR- amplified fragment relative to the circular plasmid in this experimental model, while other factors apparently have a lesser impact. At the organism level, no significant difference in the expression efficiency of the plasmid and PCR- amplified fragment has been revealed. Comparison of the vector efficiencies in in vivo and in vitro systems demonstrates that the level of luciferase in the D . rerio cell lysate, normalized to the molar concentration of the vector, is by three orders of magnitude higher than that after the cell transfection in vitro , which indicates that the quantitative data obtained for in vitro systems should not be directly extrapolated to the organism level.