Functional efficiency of PCR vectors in vitro and at the organism level

Abstract

The functional efficiency of the expression cassettes integrated into a plasmid and a PCRamplified 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.

Transfection of Sponge Cells and Intracellular Localization of Cancer-Related MYC, RRAS2, and DRG1 Proteins

“…Transfection is also efficient in other vertebrate cell lines and cells, for example, by the microinjection of foreign DNA into Xenopus laevis oocytes [ 13 , 14 ]. In addition, an efficient transfection protocol was developed for the zebrafish cell lines, Pac2, ZF4, and ZFL, by chemical transfection [ 15 , 16 , 17 , 18 , 19 , 20 ] or cassette-based transfection [ 21 ]. Although there are fewer cell lines derived from different invertebrate taxa than vertebrates, there are more than 500 cell lines derived from insects.…”

Efficiency of Promoters of Human Genes FAP and CTGF at Organism Level in a Danio rerio Model

“…High fecundity and rapid development relative to other organism models make zebrafish a convenient organism-level screening system. As demonstrated previously, the zebrafish model makes it possible to evaluate the basic properties of vector systems such as time-related accumulation and tissue distribution of the target protein as well as tissue-specificity and toxicity of the vector and target expression product [ 33 , 34 , 35 ], which substantiated the use of this model system to evaluate the expression efficiency of the promoter elements. Here, we used the model of developing zebrafish embryos to comparatively analyze the efficiency of human FAP and CTGF promoters as well as that of immediate early genes of Human cytomegalovirus (CMV) in nonviral vectors with Photinus pyralis luciferase gene and enhanced green fluorescent protein (EGFP) gene as reporter ones.…”

“…This can result from the effect of zebrafish-specific transcription factors on the function of studied expression constructs, and it should be considered when using this model. Nevertheless, this developing zebrafish model can provide new data on the vector function at the organism level and could be employed to compare the efficiency of human FAP and CTGF promoters in a multi-population system [ 33 , 34 , 35 ].…”

Embryotoxic activity of 3C protease of human hepatitis A virus in developing Danio rerio embryos

“…Vector constructs. The expression vector pCI (Promega, United States) and the derived genetic constructs pCI-3Cpro, pCI-3Cmut, pCI-luc were used; the latter was constructed previously 20 . The proteolytic activity of 3Cpro was analyzed using pGloSensor-30F-LRTQS.…”

“…www.nature.com/scientificreports/ Validation of expression activity of genetic constructs. The expression efficiency of pCI-luc was described previously 20 . The capacity of the generated vectors to synthesize the target proteins was evaluated by transfecting HEK293 cells with pCI-3Cpro and pCI-3Cmut.…”