Functional efficiency of PCR vectors in vitro and at the organism level
| Autor: | Sergey V. Kostrov, Ilya V. Demidyuk, Eugene D. Sverdlov, Maria A. Karaseva, D. R. Safina, Alexey A. Komissarov, Polina I. Selina, Marina P. Roschina |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Embryology Molecular biology Artificial Gene Amplification and Extension Efficiency Polymerase Chain Reaction Biochemistry law.invention 0302 clinical medicine Plasmid law Genes Reporter Transgenes Luciferases Promoter Regions Genetic Polymerase chain reaction Zebrafish Multidisciplinary biology Chemistry Eukaryota Animal Models Enzymes Experimental Organism Systems Osteichthyes 030220 oncology & carcinogenesis Vertebrates Medicine Expression cassette Oxidoreductases Luciferase Plasmids Research Article Substitution Mutation Science Genetic Vectors Green Fluorescent Proteins DNA construction Transfection 03 medical and health sciences Model Organisms Cell Line Tumor Photinus pyralis Genetics Animals Point Mutation Gene Reporter gene Embryos Fireflies Organisms Biology and Life Sciences Proteins biology.organism_classification In vitro Research and analysis methods 030104 developmental biology Molecular biology techniques Fish Plasmid Construction Mutation Enzymology Animal Studies Developmental Biology |
| Zdroj: | PLoS ONE PLoS ONE, Vol 15, Iss 4, p e0232045 (2020) |
| ISSN: | 1932-6203 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
| Nepřihlášeným uživatelům se plný text nezobrazuje | K zobrazení výsledku je třeba se přihlásit. |