Quantitation of Transient Gene Expression After Electroporation

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 1992 Jun 25

PMID 1620612

Citations 5

Authors

M K Showe

D L Williams

L C Showe

Affiliations

Soon will be listed here.

Abstract

The combination of a photometric reporter-gene assay, with transfection by electroporation, is potentially a rapid and sensitive tool for the study of genetic regulatory elements in many types of cells. We have found that the sensitivity, accuracy, and reproducibility of the technique is greatly improved by the inclusion of appropriately chosen carrier DNA as the primary DNA species present during electroporation. By using high levels of carrier, the activities of constructs of differing sizes can be quantitatively compared, active constructs can be assayed with sub-microgram amounts of plasmid, and the activities of the constructs are linear over a wide concentration of DNA. In addition, the activity of miniprep DNA can be screened without purification on CsCl gradients giving activities equal to CsCl-purified DNA. This is extremely useful when doing preliminary screening of large numbers of constructs for promoter or enhancer activities. We report the results of testing various types of DNA as carrier, and the parameters for optimizing its use.

Citing Articles

Immunotherapeutic suppression of indoleamine 2,3-dioxygenase and tumor growth with ethyl pyruvate.

Muller A, DuHadaway J, Jaller D, Curtis P, Metz R, Prendergast G Cancer Res. 2010; 70(5):1845-53.

PMID: 20160032 PMC: 3176890. DOI: 10.1158/0008-5472.CAN-09-3613.

Efficient transcription of the human angiotensin II type 2 receptor gene requires intronic sequence elements.

Warnecke C, Willich T, Holzmeister J, Bottari S, Fleck E, Regitz-Zagrosek V Biochem J. 1999; 340 ( Pt 1):17-24.

PMID: 10229654 PMC: 1220217.

Shared promoter elements between a viral superantigen and the major histocompatibility complex class II-associated invariant chain.

Arroyo J, Winchester E, McLellan B, Huber B J Virol. 1997; 71(2):1237-45.

PMID: 8995647 PMC: 191178. DOI: 10.1128/JVI.71.2.1237-1245.1997.

A luciferase expression system for Physarum that facilitates analysis of regulatory elements.

Bailey J, Benard M, Burland T Curr Genet. 1994; 26(2):126-31.

PMID: 8001165 DOI: 10.1007/BF00313799.

A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.

Boussif O, Lezoualch F, Zanta M, Mergny M, Scherman D, Demeneix B Proc Natl Acad Sci U S A. 1995; 92(16):7297-301.

PMID: 7638184 PMC: 41326. DOI: 10.1073/pnas.92.16.7297.

References

Anderson M, Spandidos D, Coggins J . Electroporation of lymphoid cells: factors affecting the efficiency of transfection. J Biochem Biophys Methods. 1991; 22(3):207-22. DOI: 10.1016/0165-022x(91)90069-9. View

Beug H, Palmieri S, Freudenstein C, Zentgraf H, Graf T . Hormone-dependent terminal differentiation in vitro of chicken erythroleukemia cells transformed by ts mutants of avian erythroblastosis virus. Cell. 1982; 28(4):907-19. DOI: 10.1016/0092-8674(82)90070-8. View

Chu G, Hayakawa H, Berg P . Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res. 1987; 15(3):1311-26. PMC: 340526. DOI: 10.1093/nar/15.3.1311. View

DE Wet J, Wood K, DeLuca M, Helinski D, Subramani S . Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987; 7(2):725-37. PMC: 365129. DOI: 10.1128/mcb.7.2.725-737.1987. View

Rosenthal N . Identification of regulatory elements of cloned genes with functional assays. Methods Enzymol. 1987; 152:704-20. DOI: 10.1016/0076-6879(87)52075-4. View

Loh E, Lanier L, Turck C, Littman D, Davis M, Chien Y . Identification and sequence of a fourth human T cell antigen receptor chain. Nature. 1987; 330(6148):569-72. DOI: 10.1038/330569a0. View

Weiss A, Newton M, Crommie D . Expression of T3 in association with a molecule distinct from the T-cell antigen receptor heterodimer. Proc Natl Acad Sci U S A. 1986; 83(18):6998-7002. PMC: 386639. DOI: 10.1073/pnas.83.18.6998. View

An G, Hidaka K, Siminovitch L . Expression of bacterial beta-galactosidase in animal cells. Mol Cell Biol. 1982; 2(12):1628-32. PMC: 369970. DOI: 10.1128/mcb.2.12.1628-1632.1982. View

Andreason G, Evans G . Introduction and expression of DNA molecules in eukaryotic cells by electroporation. Biotechniques. 1988; 6(7):650-60. View

10.

Harvey R, Martinerie C, Sun L, Williams D, Showe L, Marteneire C . Translocations and rearrangements in T-cell acute leukemias with the t(11;14) (p13;q11) chromosomal translocations. Oncogene. 1989; 4(3):341-9. View

11.

Ho I, Yang L, Morle G, Leiden J . A T-cell-specific transcriptional enhancer element 3' of C alpha in the human T-cell receptor alpha locus. Proc Natl Acad Sci U S A. 1989; 86(17):6714-8. PMC: 297916. DOI: 10.1073/pnas.86.17.6714. View

12.

McNally M, Lebkowski J, Okarma T, Lerch L . Optimizing electroporation parameters for a variety of human hematopoietic cell lines. Biotechniques. 1988; 6(9):882-6. View

13.

Redondo J, Hata S, Brocklehurst C, Krangel M . A T cell-specific transcriptional enhancer within the human T cell receptor delta locus. Science. 1990; 247(4947):1225-9. DOI: 10.1126/science.2156339. View

14.

Cioe L, McNab A, Hubbell H, Meo P, Curtis P, Rovera G . Differential expression of the globin genes in human leukemia K562(S) cells induced to differentiate by hemin or butyric acid. Cancer Res. 1981; 41(1):237-43. View

15.

Hall C, Jacob P, Ringold G, Lee F . Expression and regulation of Escherichia coli lacZ gene fusions in mammalian cells. J Mol Appl Genet. 1983; 2(1):101-9. View

16.

Borras T, Peterson C, Piatigorsky J . Evidence for positive and negative regulation in the promoter of the chicken delta 1-crystallin gene. Dev Biol. 1988; 127(1):209-19. DOI: 10.1016/0012-1606(88)90202-3. View