Accelerated assessing of antisense RNA efficacy using a chimeric enhanced green fluorescent protein-antisense RNA-producing vector.
The selection of suitable parts of a gene as antisense RNA sequences is largely a matter of trial and error and, as a consequence, a rather time-consuming process. In this study, we present a rapid and reproducible method to bypass this protracted procedure by using a chimeric enhanced green fluorescent protein (EGFP)-antisense RNA-producing vector. The combination of a reporter gene and antisense RNA allows easy measurement by flow cytometry of antisense RNA efficacy in successfully transfected cells shortly after transfection. Four chimeric EGFP-p185c-erbB-2-antisense RNA vectors were constructed and transfected into the p185-c-erbB-2-overexpressing cell line SKBR3. Within 1 week, we were able to estimate the inhibitory capacities of the different antisense RNA sequences used in this study. Our results strongly suggest that a chimeric EGFP-antisense RNA vector is an appropriate tool to expedite the laboratory work and time in screening the efficacy of antisense RNA strategies.
Dittmar
T
T
Institute of Immunology, University of Witten/Herdecke, Germany.
Schäfer
F
F
Brandt
B H
BH
Zänker
K S
KS
D002455:Cell Division; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics*; D018734:Genes, erbB-2 / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016372:RNA, Antisense / Q000235:genetics* / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics; D015203:Reproducibility of Results; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D016372:RNA, Antisense; D018719:Erb-b2 Receptor Tyrosine Kinases