Gene transfer: DNA microinjection compared with DNA transfection with a very high efficiency

Y. M. Shen, R. R. Hirschhorn, W. E. Mercer, E. Surmacz, Y. Tsutsui, K. J. Soprano, R. Baserga

Research output: Contribution to journalArticle

Abstract

We have developed a procedure that gives a very high efficiency of transfection in mammalian cells with low-molecular-weight DNA (~10 4 base pairs). The procedure uses cells in suspension that are shocked with polyethylene glycol 4 h after replating. We compared this transfection technique to the standard technique involving manual microinjection of DNA into the nuclei of mammalian cells, using recombinant plasmids containing the simian virus 40 A gene or the herpes simplex virus thymidine kinase gene or both. The efficiency of transfection depends on a number of variables, the most important of which is the difference in transfectability of different cell lines. In our laboratory, the cell line that had the highest efficiency of transfection was tk -ts13, which is derived from baby hamster kidney cells that are deficient in thymidine kinase and temperature sensitive for growth. Under the appropriate conditions, as many as 70% of these cells can be transfected so that transient gene expression can be detected. With the manual microinjection technique, gene expression is independent of the cell line used and occurs faster than after transfection. The results suggest that the critical stage in transfection is the delivery of DNA molecules to the nucleus. Our experiments also indicate that an enzymatic function, in our case, thymidine kinase activity, gives a higher percentage of positive transfectants than when proteins are visualized only by indirect immunofluorescence. The transfection procedure described in this paper is simple and reproducible and, although less efficient than microinjection, ought to be useful in phenotypic and genotypic studies in which transfer of genes to a large number of cells is desirable.

Original languageEnglish (US)
Pages (from-to)1145-1154
Number of pages10
JournalMolecular and Cellular Biology
Volume2
Issue number9
StatePublished - 1982
Externally publishedYes

Fingerprint

Microinjections
Transfection
DNA
Thymidine Kinase
Genes
Cell Line
Gene Expression
Simian virus 40
Simplexvirus
Indirect Fluorescent Antibody Technique
Cell Nucleus
Base Pairing
Cricetinae
Suspensions
Plasmids
Cell Count
Molecular Weight
Kidney
Temperature
Growth

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Shen, Y. M., Hirschhorn, R. R., Mercer, W. E., Surmacz, E., Tsutsui, Y., Soprano, K. J., & Baserga, R. (1982). Gene transfer: DNA microinjection compared with DNA transfection with a very high efficiency. Molecular and Cellular Biology, 2(9), 1145-1154.

Gene transfer : DNA microinjection compared with DNA transfection with a very high efficiency. / Shen, Y. M.; Hirschhorn, R. R.; Mercer, W. E.; Surmacz, E.; Tsutsui, Y.; Soprano, K. J.; Baserga, R.

In: Molecular and Cellular Biology, Vol. 2, No. 9, 1982, p. 1145-1154.

Research output: Contribution to journalArticle

Shen, YM, Hirschhorn, RR, Mercer, WE, Surmacz, E, Tsutsui, Y, Soprano, KJ & Baserga, R 1982, 'Gene transfer: DNA microinjection compared with DNA transfection with a very high efficiency', Molecular and Cellular Biology, vol. 2, no. 9, pp. 1145-1154.
Shen YM, Hirschhorn RR, Mercer WE, Surmacz E, Tsutsui Y, Soprano KJ et al. Gene transfer: DNA microinjection compared with DNA transfection with a very high efficiency. Molecular and Cellular Biology. 1982;2(9):1145-1154.
Shen, Y. M. ; Hirschhorn, R. R. ; Mercer, W. E. ; Surmacz, E. ; Tsutsui, Y. ; Soprano, K. J. ; Baserga, R. / Gene transfer : DNA microinjection compared with DNA transfection with a very high efficiency. In: Molecular and Cellular Biology. 1982 ; Vol. 2, No. 9. pp. 1145-1154.
@article{b368decf8d05453b90a6916606264a50,
title = "Gene transfer: DNA microinjection compared with DNA transfection with a very high efficiency",
abstract = "We have developed a procedure that gives a very high efficiency of transfection in mammalian cells with low-molecular-weight DNA (~10 4 base pairs). The procedure uses cells in suspension that are shocked with polyethylene glycol 4 h after replating. We compared this transfection technique to the standard technique involving manual microinjection of DNA into the nuclei of mammalian cells, using recombinant plasmids containing the simian virus 40 A gene or the herpes simplex virus thymidine kinase gene or both. The efficiency of transfection depends on a number of variables, the most important of which is the difference in transfectability of different cell lines. In our laboratory, the cell line that had the highest efficiency of transfection was tk -ts13, which is derived from baby hamster kidney cells that are deficient in thymidine kinase and temperature sensitive for growth. Under the appropriate conditions, as many as 70{\%} of these cells can be transfected so that transient gene expression can be detected. With the manual microinjection technique, gene expression is independent of the cell line used and occurs faster than after transfection. The results suggest that the critical stage in transfection is the delivery of DNA molecules to the nucleus. Our experiments also indicate that an enzymatic function, in our case, thymidine kinase activity, gives a higher percentage of positive transfectants than when proteins are visualized only by indirect immunofluorescence. The transfection procedure described in this paper is simple and reproducible and, although less efficient than microinjection, ought to be useful in phenotypic and genotypic studies in which transfer of genes to a large number of cells is desirable.",
author = "Shen, {Y. M.} and Hirschhorn, {R. R.} and Mercer, {W. E.} and E. Surmacz and Y. Tsutsui and Soprano, {K. J.} and R. Baserga",
year = "1982",
language = "English (US)",
volume = "2",
pages = "1145--1154",
journal = "Molecular and Cellular Biology",
issn = "0270-7306",
publisher = "American Society for Microbiology",
number = "9",

}

TY - JOUR

T1 - Gene transfer

T2 - DNA microinjection compared with DNA transfection with a very high efficiency

AU - Shen, Y. M.

AU - Hirschhorn, R. R.

AU - Mercer, W. E.

AU - Surmacz, E.

AU - Tsutsui, Y.

AU - Soprano, K. J.

AU - Baserga, R.

PY - 1982

Y1 - 1982

N2 - We have developed a procedure that gives a very high efficiency of transfection in mammalian cells with low-molecular-weight DNA (~10 4 base pairs). The procedure uses cells in suspension that are shocked with polyethylene glycol 4 h after replating. We compared this transfection technique to the standard technique involving manual microinjection of DNA into the nuclei of mammalian cells, using recombinant plasmids containing the simian virus 40 A gene or the herpes simplex virus thymidine kinase gene or both. The efficiency of transfection depends on a number of variables, the most important of which is the difference in transfectability of different cell lines. In our laboratory, the cell line that had the highest efficiency of transfection was tk -ts13, which is derived from baby hamster kidney cells that are deficient in thymidine kinase and temperature sensitive for growth. Under the appropriate conditions, as many as 70% of these cells can be transfected so that transient gene expression can be detected. With the manual microinjection technique, gene expression is independent of the cell line used and occurs faster than after transfection. The results suggest that the critical stage in transfection is the delivery of DNA molecules to the nucleus. Our experiments also indicate that an enzymatic function, in our case, thymidine kinase activity, gives a higher percentage of positive transfectants than when proteins are visualized only by indirect immunofluorescence. The transfection procedure described in this paper is simple and reproducible and, although less efficient than microinjection, ought to be useful in phenotypic and genotypic studies in which transfer of genes to a large number of cells is desirable.

AB - We have developed a procedure that gives a very high efficiency of transfection in mammalian cells with low-molecular-weight DNA (~10 4 base pairs). The procedure uses cells in suspension that are shocked with polyethylene glycol 4 h after replating. We compared this transfection technique to the standard technique involving manual microinjection of DNA into the nuclei of mammalian cells, using recombinant plasmids containing the simian virus 40 A gene or the herpes simplex virus thymidine kinase gene or both. The efficiency of transfection depends on a number of variables, the most important of which is the difference in transfectability of different cell lines. In our laboratory, the cell line that had the highest efficiency of transfection was tk -ts13, which is derived from baby hamster kidney cells that are deficient in thymidine kinase and temperature sensitive for growth. Under the appropriate conditions, as many as 70% of these cells can be transfected so that transient gene expression can be detected. With the manual microinjection technique, gene expression is independent of the cell line used and occurs faster than after transfection. The results suggest that the critical stage in transfection is the delivery of DNA molecules to the nucleus. Our experiments also indicate that an enzymatic function, in our case, thymidine kinase activity, gives a higher percentage of positive transfectants than when proteins are visualized only by indirect immunofluorescence. The transfection procedure described in this paper is simple and reproducible and, although less efficient than microinjection, ought to be useful in phenotypic and genotypic studies in which transfer of genes to a large number of cells is desirable.

UR - http://www.scopus.com/inward/record.url?scp=0020376744&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0020376744&partnerID=8YFLogxK

M3 - Article

C2 - 6294505

AN - SCOPUS:0020376744

VL - 2

SP - 1145

EP - 1154

JO - Molecular and Cellular Biology

JF - Molecular and Cellular Biology

SN - 0270-7306

IS - 9

ER -