Nucleic Acids Research, Vol. 18, No. 10 3079
.) 1990 Oxford University Press
Mini-transfections on 96-well microtiter plates Giovanna Buttice and Markku Kurkinen* Department of Medicine, University of Medicine and Dentistry of New Jersey - Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA Submitted April 11, 1990
Transient transfection is one of the most commonly used techniques to study gene regulation. Different reporter genes have been used to quantitate the promoter activity. CAT (chloramphenicol acetyltransferase) is the reporter gene most often used in these experiments (1). For increased sensitivity, growth hormone and luciferase have also been used as reporter genes (2, 3). For a rapid analysis of gene expression, we describe here a fast and convenient transfection protocol for growth hormone reporter gene. Because growth hormone is secreted, we have scaled down the number of transfected cells so that cell cultures on 96-well microtiter plates are adequate for routine work. Using this transfection protocol, amounts of DNA, chemicals and nutrients are similarly reduced. The protocol is convenient for multi-variable studies and should facilitate screening for factors and treatments that can result in synergistic stimulation or repression of gene activity. As shown in Fig. 1, results from the mini-transfections are compatible with those obtained by large scale CAT assay or RNase protection technique.
1 RNase
3 20
2.4
CAT .Addffs,6. 41P
i
4UUU,J
30000
ACKNOWLEDGEMENT This work was supported by the NIH.
2
CL c)
GH-i
5.9
20000
REFERENCES 10000
1. Gorman,C.M., Moffat,L.F. and Howard,B.H. (1982) Mol. Cell. Biol. 2,
1044-1051. 2. Selden,R.F., Burke-Howie,K., Rowe,M.E., Goodman,H.M. and Moore,D.D. (1986) Mol. Cell. Biol. 6, 3173-3179. 3. De Wet,J.R., Wood,K.V., DeLuca,M., Helinski,D.R. and Subramani,S. (1987) Mol. Cell. Biol. 7, 725-737. 4. Ausubel,M.F., Brent,R., Kingston,R.E., Moore,D.D., Seidman,J.G., Smith,J.A. and Struhl,K. (1988) Current Protocols in Molecular Biology, John Wiley & Sons, New York.
*
To whom correspondence should be addressed
Vector Control
TPA
Figure 1. TPA-induction of stromelysin transcription. Lanes show the activity for promoterless reporter plasmid (1), basal level (2) and TPA-induced (3) stromelysin promoter activity measured by RNase protection, CAT, or growth hormone assay. The RNase protected band (79 nucleotides) demonstrates correct transcription initiation from the transfected stromelysin promoter. To calculate fold-induction (numbers on lane 3) the promoterless activity has been subtracted from experimental values. In the RNase protection experiment shown here growth hormone assay from the same plate gave 19-fold induction by TPA. HepG2 cells (HeLa cells work as well) are plated on 96-well plate at density of 104 cells/well. Transfection per well consists of 4 IL (50 ng) of growth hormone plasmid with human stromelysin promoter (-1303 to +4), 4 1l of Buffer A, 8 1l of Buffer B, mixed and incubated as described in Cell-transphect kit (Pharmacia), mixed with 84 tl of DMEM containing 10% FCS, and added into the well. Twelve hours later cells are rinsed twice, incubated for 2 days in 50 1I of medium with or without TPA (100 ng/ml) and 20 yd is assayed for secreted growth hormone using a radioimmunoassay kit (Nichols Institute). For CAT assay or RNase protection assay, 10 ,ug of CAT or growth hormone plasmid, respectively, with the stromelysin promoter were transfected into 106 cells/100 mm dish and treated as above. CAT assay and RNase protection assay were performed as described (4).
.) 1990 Oxford University Press
Mini-transfections on 96-well microtiter plates Giovanna Buttice and Markku Kurkinen* Department of Medicine, University of Medicine and Dentistry of New Jersey - Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA Submitted April 11, 1990
Transient transfection is one of the most commonly used techniques to study gene regulation. Different reporter genes have been used to quantitate the promoter activity. CAT (chloramphenicol acetyltransferase) is the reporter gene most often used in these experiments (1). For increased sensitivity, growth hormone and luciferase have also been used as reporter genes (2, 3). For a rapid analysis of gene expression, we describe here a fast and convenient transfection protocol for growth hormone reporter gene. Because growth hormone is secreted, we have scaled down the number of transfected cells so that cell cultures on 96-well microtiter plates are adequate for routine work. Using this transfection protocol, amounts of DNA, chemicals and nutrients are similarly reduced. The protocol is convenient for multi-variable studies and should facilitate screening for factors and treatments that can result in synergistic stimulation or repression of gene activity. As shown in Fig. 1, results from the mini-transfections are compatible with those obtained by large scale CAT assay or RNase protection technique.
1 RNase
3 20
2.4
CAT .Addffs,6. 41P
i
4UUU,J
30000
ACKNOWLEDGEMENT This work was supported by the NIH.
2
CL c)
GH-i
5.9
20000
REFERENCES 10000
1. Gorman,C.M., Moffat,L.F. and Howard,B.H. (1982) Mol. Cell. Biol. 2,
1044-1051. 2. Selden,R.F., Burke-Howie,K., Rowe,M.E., Goodman,H.M. and Moore,D.D. (1986) Mol. Cell. Biol. 6, 3173-3179. 3. De Wet,J.R., Wood,K.V., DeLuca,M., Helinski,D.R. and Subramani,S. (1987) Mol. Cell. Biol. 7, 725-737. 4. Ausubel,M.F., Brent,R., Kingston,R.E., Moore,D.D., Seidman,J.G., Smith,J.A. and Struhl,K. (1988) Current Protocols in Molecular Biology, John Wiley & Sons, New York.
*
To whom correspondence should be addressed
Vector Control
TPA
Figure 1. TPA-induction of stromelysin transcription. Lanes show the activity for promoterless reporter plasmid (1), basal level (2) and TPA-induced (3) stromelysin promoter activity measured by RNase protection, CAT, or growth hormone assay. The RNase protected band (79 nucleotides) demonstrates correct transcription initiation from the transfected stromelysin promoter. To calculate fold-induction (numbers on lane 3) the promoterless activity has been subtracted from experimental values. In the RNase protection experiment shown here growth hormone assay from the same plate gave 19-fold induction by TPA. HepG2 cells (HeLa cells work as well) are plated on 96-well plate at density of 104 cells/well. Transfection per well consists of 4 IL (50 ng) of growth hormone plasmid with human stromelysin promoter (-1303 to +4), 4 1l of Buffer A, 8 1l of Buffer B, mixed and incubated as described in Cell-transphect kit (Pharmacia), mixed with 84 tl of DMEM containing 10% FCS, and added into the well. Twelve hours later cells are rinsed twice, incubated for 2 days in 50 1I of medium with or without TPA (100 ng/ml) and 20 yd is assayed for secreted growth hormone using a radioimmunoassay kit (Nichols Institute). For CAT assay or RNase protection assay, 10 ,ug of CAT or growth hormone plasmid, respectively, with the stromelysin promoter were transfected into 106 cells/100 mm dish and treated as above. CAT assay and RNase protection assay were performed as described (4).
