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Gene Delivery into Neuronal Cells by Calcium Phosphate-Mediated Transfection
METHODS: A Companion to Methods in Enzymology 10, 289–291 (1996) Article No. 0105
Gene Delivery into Neuronal Cells by Calcium Phosphate-Mediated Transfection Andrea Watson1 and David Latchman2 Department of Molecular Pathology, University College London Medical School, The Windeyer Building, Cleveland Street, London W1P 6DB, United Kingdom
This paper describes a method for introducing DNA constructs into primary adult dorsal root ganglion (DRG) neuron cultures. The method uses a modified calcium phosphate technique and enables relatively small numbers of cells to be used. We have used this method to study the promoter sequences responsible for mediating gene activity and nerve growth factor responsiveness in DRG neurons. It can also be used, however, for other purposes such as testing the effect on neuronal function of overexpressing a specific gene product. q 1996 Academic Press, Inc.
Once a gene has been shown to be regulated at the level of transcription, the next step is to map the elements within the promoter of the gene that are responsible for expression. To test promoter activity, putative regulatory DNA sequences from the 5* region of the gene of interest are linked to a gene encoding a readily assayable product, such as chloramphenicol acetyl transferase, b-galactosidase (b-gal), or luciferase, and this construct is introduced into cells; a number of plasmid vectors containing the coding regions of these genes downstream of a multiple cloning site are now commercially available. The effect of the regulatory sequences on the production of the assayable product can then be assessed (1). There has been much interest in recent years in the tissue-specific regulation of gene expression resulting in the expression of specific genes in one particular cell type only. Thus for example, the regulation of a variety of genes such as the SCG 10 gene and the calcitonin/ calcitonin gene-related peptide (CGRP) gene in a neuronal cell-specific manner has been described in several papers (2, 3). In addition the processes mediating the 1
Present address: Eisai London Research Laboratories, Bernard Katz Building, University College London, Gower Street, London WC1E 6BT. 2 To whom correspondence should be addressed. Fax: 44-171-3873310.
activation of a specific gene by a particular inducer may differ in different cell types and even between different neuronal cells. For instance, we have recently demonstrated that the mechanisms mediating nerve growth factor (NGF) inducibility of the CGRP gene differ in the dorsal root ganglion (DRG) and the PC12 pheochromocytoma cell line (4). Data such as these highlight the necessity to perform experiments like promoter mapping in the appropriate cells that naturally express the gene under investigation. A number of methods for introducing DNA constructs into cell lines using transfection have been described. However, these are usually performed on cultured cell lines and require large amounts of material (Ç106 cells) (5, 6). In the case of DRG it is difficult to obtain these amounts of homogeneous material to enable the use of existing methods, and the cell lines that have been derived from DRGs do not respond to NGF. We have therefore developed a method for transfecting small numbers of DRG sensory neurons using a modified calcium phosphate technique that will achieve transfection efficiencies of between 10 and 15% of cells in the culture.
METHODS DRG Cultures Purified cultures of adult rat DRG neurons were obtained as described in Ref. 7. The day before transfection 1.5 1 105 neurons were plated onto poly-L-lysinecoated 35-mm dishes. Cells were grown in F-14 medium containing 4% Ultraser G (Gibco), 10 mM ara-C, 5000 units penicillin/5000 units streptomycin, in an atmosphere of 3% CO2 . DNA To obtain the highest transfection efficiencies plasmid DNA was purified either using PEG precipitation or by double-banding on cesium chloride density gradi289
1046-2023/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
AID
Methods 334Z
/
6709$$$$21
09-24-96 14:04:08
metha
AP: Methods
WATSON AND LATCHMAN
290
ents. DNA that was to be used for transfection was resuspended in sterile water, as a large volume of added DNA in Tris buffer may change the pH and efficient transfection occurs in a very narrow pH range. Transfection 1. Following the overnight incubation the medium was replaced with 0.9 ml fresh medium containing 10 mg/ml polyornithine (diluted from a 10 mg/ml stock) (8), and the cells were returned to the incubator for approximately 1 h to allow the medium to equilibrate. 2. The calcium phosphate DNA precipitate was prepared under sterile conditions as described below; all reagents were at room temperature before the precipitate was made. In tube A: 10 mg DNA 6.2 ml 2
M
CaCl2
ddH2O to a final volume of 50 ml In tube B: 50 ml 21 Hepes-buffered saline (HBS). This was diluted from a 101 stock that contains 8.18% NaCl (w/v), 5.94% Hepes (w/v), 0.2% Na2HPO4r2H2O (w/v). Prior to transfection experiments the 101 stock was diluted to a 21 working solution with ddH2O and adjusted to pH 7.12 using 0.4 M NaOH. It may be necessary to make up several batches of HBS over a pH range 7 to 7.2 and test each batch for the quality of precipitate and for the efficiency of transfection. Once a particular batch had been tested it was stored at 0207C and reproducible results could be obtained over a long period of time. 3. Tube B was vortexed, with the speed adjusted such that the tube could be vortexed with the cap off. The contents of tube A were added dropwise to the HBS while continuing to vortex. The resulting solution should be translucent in appearance due to the formation of a fine coprecipitate of DNA with calcium and phosphate. If the solution appeared opaque then it was discarded. The precipitate was left for 30 min before being added to the medium dropwise. The cells were returned to the incubator immediately following addition of the DNA to ensure that the pH did not change. 4. The cells were incubated for 7 h and then the transfection medium was removed and the cells ‘‘shocked’’ by treatment with 30% DMSO in serum-free medium for 3 min at room temperature. The cells were then washed thoroughly, with care taken not to remove all the medium, before the addition of 2 ml medium and the return to the incubator. The addition of DMSO led to some cell death (around 10–20% of the cells, as assessed by trypan blue exclusion) but increased transfection efficiency 3- to 5-fold. The precise mecha-
AID
Methods 334Z
/
6709$$$$22
nism of action of DMSO is unknown but it may modify cell membrane structure to enhance uptake of DNA. For experiments in which the effect of a growth factor on the transfected DNA was being studied, growth factor was added to the medium following transfection. 5. Cells were incubated for a further 48 h to allow expression of the reporter gene. Cells were then harvested by applying a stream of medium from a 1-ml Gilson tip to the tissue culture dish so that the neuronal network was removed while any contaminating nonneuronal cells were left on the dish. To determine transfection efficiency, cells were fixed at the end of the experiment and were stained for the protein product of the reporter gene; this was done using either histochemical or immunostaining techniques (9). 6. Extracts were balanced for protein as determined by the method of Bradford (10). Any variation in plasmid DNA uptake between samples was accounted for by slot blotting 15th of the extract and probing for transfected DNA using a probe derived from the vector DNA (11). Differences in the efficiency of DNA uptake between cells transfected with different plasmids are usually controlled for by cotransfecting cells with plasmid DNA containing a control promoter driving the expression of a reporter gene encoding a different assayable product; extracts are then assayed for both reporter genes. However, due to the small numbers of cells obtained from primary DRG cultures, there was not enough material to carry out more than one assay per experimental construct, hence extracts were standardized by plasmid DNA uptake. 7. Promoter activity was determined by assaying the activity of the enzyme encoded by the plasmid DNA. The activity of the enzyme provides a measure of the promoter activity under various conditions. Once a region of the promoter that conferred a pattern of regulation had been identified, the promoter was truncated until the effect was lost, allowing identification of the precise region of the promoter that conferred either basal activity or growth factor induction to be determined. It is also possible to take other approaches to promoter analysis using this technique. For example, expression vectors containing the cDNA for a known transcription factor that may bind the promoter of interest can be cotransfected along with the putative promoter sequence linked upstream of a reporter gene. If the transcription factor is able to modify the promoter activity then there will be a change in the level of reporter gene expression.
CONCLUSION This method provides a quick and reliable method for transient transfection of adult DRG neurons and
09-24-96 14:04:08
metha
AP: Methods
TRANSFECTION OF DRG NEURONS
it requires no special equipment. We have initially used this technique to look at regulation of the CGRP promoter by NGF in DRG neurons (4, 12). In addition to the study of promoter regulation, it can be used to study the effect of overexpressing specific genes in neuronal cells. Thus the transfected cells in the culture can be detected by cotransfecting the gene of interest with a marker gene such as b-galactosidase or alkaline phosphatase. Any phenotype produced by overexpressing the gene of interest can be assessed by comparing the phenotype of transfected cells stained with the marker gene with untransfected, unstained cells in the culture. Consequently, with the advent of improved means to mark transfected cells such as Green Fluorescent Protein (13), many of the experiments that were previously possible only using microinjection techniques should now be able to be performed using transfection. Although the work described in this paper describes transfection of DRG neurons, there is no reason why modifications of this technique should not be applicable to the transfection of other primary neuronal cultures.
AID
Methods 334Z
/
6709$$$$22
291
REFERENCES 1. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Vol. 2, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2. Stolarsky-Fredman, L., Leff, S. E., Klein, E. S., Crenshaw, E. B., Yeakley, J., and Rosenfeld, M. G. (1990) Mol. Endocrinol. 4, 497–504. 3. Kraner, S. D., Chong, J. A., Tsay, H. J., and Mandel, G. (1992) Neuron 9, 37–44. 4. Watson, A., Ensor, E., Symes, A., Winter, J., Kendall, G., and Latchman, D. (1995) Eur. J. Neurosci. 7, 394–400. 5. Gorman, C. (1985) DNA Cloning: A Practical Approach (Glover, D. M., Ed.), Vol. 2, pp. 143–190, IRL Press, Eynsham, England. 6. Graham, F. I., and van der Eb, A. J. (1973) Virology 52, 456– 457. 7. Lindsay, R. M. (1988) J. Neurosci. 8, 2394–2405. 8. Dong, Y., Skoultchi, A. I., and Pollard, J. W. (1993) Nucleic Acids Res. 21, 771–772. 9. Lim, K., and Chae, C. B. (1989) BioTechniques 7, 576–579. 10. Bradford, M. (1976) Anal. Biochem. 72, 248–254. 11. Abken, J., and Reifenrath, B. (1992) Nucleic Acids Res. 20, 3527. 12. Watson, A., and Latchman, D. S. (1995) J. Biol. Chem. 270, 9655–9660. 13. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994) Science 263, 802–805.
09-24-96 14:04:08
metha
AP: Methods
Gene Delivery into Neuronal Cells by Calcium Phosphate-Mediated Transfection Andrea Watson1 and David Latchman2 Department of Molecular Pathology, University College London Medical School, The Windeyer Building, Cleveland Street, London W1P 6DB, United Kingdom
This paper describes a method for introducing DNA constructs into primary adult dorsal root ganglion (DRG) neuron cultures. The method uses a modified calcium phosphate technique and enables relatively small numbers of cells to be used. We have used this method to study the promoter sequences responsible for mediating gene activity and nerve growth factor responsiveness in DRG neurons. It can also be used, however, for other purposes such as testing the effect on neuronal function of overexpressing a specific gene product. q 1996 Academic Press, Inc.
Once a gene has been shown to be regulated at the level of transcription, the next step is to map the elements within the promoter of the gene that are responsible for expression. To test promoter activity, putative regulatory DNA sequences from the 5* region of the gene of interest are linked to a gene encoding a readily assayable product, such as chloramphenicol acetyl transferase, b-galactosidase (b-gal), or luciferase, and this construct is introduced into cells; a number of plasmid vectors containing the coding regions of these genes downstream of a multiple cloning site are now commercially available. The effect of the regulatory sequences on the production of the assayable product can then be assessed (1). There has been much interest in recent years in the tissue-specific regulation of gene expression resulting in the expression of specific genes in one particular cell type only. Thus for example, the regulation of a variety of genes such as the SCG 10 gene and the calcitonin/ calcitonin gene-related peptide (CGRP) gene in a neuronal cell-specific manner has been described in several papers (2, 3). In addition the processes mediating the 1
Present address: Eisai London Research Laboratories, Bernard Katz Building, University College London, Gower Street, London WC1E 6BT. 2 To whom correspondence should be addressed. Fax: 44-171-3873310.
activation of a specific gene by a particular inducer may differ in different cell types and even between different neuronal cells. For instance, we have recently demonstrated that the mechanisms mediating nerve growth factor (NGF) inducibility of the CGRP gene differ in the dorsal root ganglion (DRG) and the PC12 pheochromocytoma cell line (4). Data such as these highlight the necessity to perform experiments like promoter mapping in the appropriate cells that naturally express the gene under investigation. A number of methods for introducing DNA constructs into cell lines using transfection have been described. However, these are usually performed on cultured cell lines and require large amounts of material (Ç106 cells) (5, 6). In the case of DRG it is difficult to obtain these amounts of homogeneous material to enable the use of existing methods, and the cell lines that have been derived from DRGs do not respond to NGF. We have therefore developed a method for transfecting small numbers of DRG sensory neurons using a modified calcium phosphate technique that will achieve transfection efficiencies of between 10 and 15% of cells in the culture.
METHODS DRG Cultures Purified cultures of adult rat DRG neurons were obtained as described in Ref. 7. The day before transfection 1.5 1 105 neurons were plated onto poly-L-lysinecoated 35-mm dishes. Cells were grown in F-14 medium containing 4% Ultraser G (Gibco), 10 mM ara-C, 5000 units penicillin/5000 units streptomycin, in an atmosphere of 3% CO2 . DNA To obtain the highest transfection efficiencies plasmid DNA was purified either using PEG precipitation or by double-banding on cesium chloride density gradi289
1046-2023/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
AID
Methods 334Z
/
6709$$$$21
09-24-96 14:04:08
metha
AP: Methods
WATSON AND LATCHMAN
290
ents. DNA that was to be used for transfection was resuspended in sterile water, as a large volume of added DNA in Tris buffer may change the pH and efficient transfection occurs in a very narrow pH range. Transfection 1. Following the overnight incubation the medium was replaced with 0.9 ml fresh medium containing 10 mg/ml polyornithine (diluted from a 10 mg/ml stock) (8), and the cells were returned to the incubator for approximately 1 h to allow the medium to equilibrate. 2. The calcium phosphate DNA precipitate was prepared under sterile conditions as described below; all reagents were at room temperature before the precipitate was made. In tube A: 10 mg DNA 6.2 ml 2
M
CaCl2
ddH2O to a final volume of 50 ml In tube B: 50 ml 21 Hepes-buffered saline (HBS). This was diluted from a 101 stock that contains 8.18% NaCl (w/v), 5.94% Hepes (w/v), 0.2% Na2HPO4r2H2O (w/v). Prior to transfection experiments the 101 stock was diluted to a 21 working solution with ddH2O and adjusted to pH 7.12 using 0.4 M NaOH. It may be necessary to make up several batches of HBS over a pH range 7 to 7.2 and test each batch for the quality of precipitate and for the efficiency of transfection. Once a particular batch had been tested it was stored at 0207C and reproducible results could be obtained over a long period of time. 3. Tube B was vortexed, with the speed adjusted such that the tube could be vortexed with the cap off. The contents of tube A were added dropwise to the HBS while continuing to vortex. The resulting solution should be translucent in appearance due to the formation of a fine coprecipitate of DNA with calcium and phosphate. If the solution appeared opaque then it was discarded. The precipitate was left for 30 min before being added to the medium dropwise. The cells were returned to the incubator immediately following addition of the DNA to ensure that the pH did not change. 4. The cells were incubated for 7 h and then the transfection medium was removed and the cells ‘‘shocked’’ by treatment with 30% DMSO in serum-free medium for 3 min at room temperature. The cells were then washed thoroughly, with care taken not to remove all the medium, before the addition of 2 ml medium and the return to the incubator. The addition of DMSO led to some cell death (around 10–20% of the cells, as assessed by trypan blue exclusion) but increased transfection efficiency 3- to 5-fold. The precise mecha-
AID
Methods 334Z
/
6709$$$$22
nism of action of DMSO is unknown but it may modify cell membrane structure to enhance uptake of DNA. For experiments in which the effect of a growth factor on the transfected DNA was being studied, growth factor was added to the medium following transfection. 5. Cells were incubated for a further 48 h to allow expression of the reporter gene. Cells were then harvested by applying a stream of medium from a 1-ml Gilson tip to the tissue culture dish so that the neuronal network was removed while any contaminating nonneuronal cells were left on the dish. To determine transfection efficiency, cells were fixed at the end of the experiment and were stained for the protein product of the reporter gene; this was done using either histochemical or immunostaining techniques (9). 6. Extracts were balanced for protein as determined by the method of Bradford (10). Any variation in plasmid DNA uptake between samples was accounted for by slot blotting 15th of the extract and probing for transfected DNA using a probe derived from the vector DNA (11). Differences in the efficiency of DNA uptake between cells transfected with different plasmids are usually controlled for by cotransfecting cells with plasmid DNA containing a control promoter driving the expression of a reporter gene encoding a different assayable product; extracts are then assayed for both reporter genes. However, due to the small numbers of cells obtained from primary DRG cultures, there was not enough material to carry out more than one assay per experimental construct, hence extracts were standardized by plasmid DNA uptake. 7. Promoter activity was determined by assaying the activity of the enzyme encoded by the plasmid DNA. The activity of the enzyme provides a measure of the promoter activity under various conditions. Once a region of the promoter that conferred a pattern of regulation had been identified, the promoter was truncated until the effect was lost, allowing identification of the precise region of the promoter that conferred either basal activity or growth factor induction to be determined. It is also possible to take other approaches to promoter analysis using this technique. For example, expression vectors containing the cDNA for a known transcription factor that may bind the promoter of interest can be cotransfected along with the putative promoter sequence linked upstream of a reporter gene. If the transcription factor is able to modify the promoter activity then there will be a change in the level of reporter gene expression.
CONCLUSION This method provides a quick and reliable method for transient transfection of adult DRG neurons and
09-24-96 14:04:08
metha
AP: Methods
TRANSFECTION OF DRG NEURONS
it requires no special equipment. We have initially used this technique to look at regulation of the CGRP promoter by NGF in DRG neurons (4, 12). In addition to the study of promoter regulation, it can be used to study the effect of overexpressing specific genes in neuronal cells. Thus the transfected cells in the culture can be detected by cotransfecting the gene of interest with a marker gene such as b-galactosidase or alkaline phosphatase. Any phenotype produced by overexpressing the gene of interest can be assessed by comparing the phenotype of transfected cells stained with the marker gene with untransfected, unstained cells in the culture. Consequently, with the advent of improved means to mark transfected cells such as Green Fluorescent Protein (13), many of the experiments that were previously possible only using microinjection techniques should now be able to be performed using transfection. Although the work described in this paper describes transfection of DRG neurons, there is no reason why modifications of this technique should not be applicable to the transfection of other primary neuronal cultures.
AID
Methods 334Z
/
6709$$$$22
291
REFERENCES 1. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Vol. 2, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2. Stolarsky-Fredman, L., Leff, S. E., Klein, E. S., Crenshaw, E. B., Yeakley, J., and Rosenfeld, M. G. (1990) Mol. Endocrinol. 4, 497–504. 3. Kraner, S. D., Chong, J. A., Tsay, H. J., and Mandel, G. (1992) Neuron 9, 37–44. 4. Watson, A., Ensor, E., Symes, A., Winter, J., Kendall, G., and Latchman, D. (1995) Eur. J. Neurosci. 7, 394–400. 5. Gorman, C. (1985) DNA Cloning: A Practical Approach (Glover, D. M., Ed.), Vol. 2, pp. 143–190, IRL Press, Eynsham, England. 6. Graham, F. I., and van der Eb, A. J. (1973) Virology 52, 456– 457. 7. Lindsay, R. M. (1988) J. Neurosci. 8, 2394–2405. 8. Dong, Y., Skoultchi, A. I., and Pollard, J. W. (1993) Nucleic Acids Res. 21, 771–772. 9. Lim, K., and Chae, C. B. (1989) BioTechniques 7, 576–579. 10. Bradford, M. (1976) Anal. Biochem. 72, 248–254. 11. Abken, J., and Reifenrath, B. (1992) Nucleic Acids Res. 20, 3527. 12. Watson, A., and Latchman, D. S. (1995) J. Biol. Chem. 270, 9655–9660. 13. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994) Science 263, 802–805.
09-24-96 14:04:08
metha
AP: Methods