- Email: [email protected]
[23] Intracellular delivery of nucleic acids and transcription factors by cationic liposomes
[23]
DELIVERY BY CATIONIC LIPOSOMES
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[23] I n t r a c e l l u l a r D e l i v e r y o f N u c l e i c A c i d s a n d Transcription Factors by Cationic Liposomes
By NEJAT D O Z G O N E ~ and PHILIP L. FELGNER Liposomes of various types have been used for the delivery of nucleic acids into cultured cells, t Earlier studies involved the encapsulation of the nucleic acids inside liposomes and the delivery of liposomes most likely via an endocytotic pathway.2 Liposomes containing the cationic lipid N-[1(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA) have been found to mediate the efficient delivery of DNA 3 and RNA4 into cells by forming a positively charged complex with the nucleic acids. Although the mechanism of delivery is not well understood, DOTMA-containing liposomes have been shown to undergo fusion with negatively charged liposomess and cells.3,6 DOTMA liposomes have also been used to deliver purified transcription factors into cells, mediating the expression of specific genes. ~ Procedures for transfection and delivery of regulatory proteins are described below. Delivery of Nucleic Acids Cells to be transfectedare plated on 60-ram diameter plastictissue culture platesat a celldensity of 0.5 × 10~/platc,and incubated overnight to become approximately 80% confluent.The transfcctionprocedure may cause toxicityin some celllinesif performed at lower celldensities.The culture medium is composed of Eagle's or Dulbccco's minimal essential medium ( D M E M ) with 10% (v/v) fctal bovine serum and Fungi-Bact solution (penicillin,streptomycin,and Fungizone; IrvineScientific,Irvine, i R. M. Straubinger and D. Papahadjopoulos, this series, Vol. 101, p. 512. : R. M. Stmubinger, K. Hong, D. S. Friend, and D. Papahadjopoulos, Cell (Cambridge, Mass.) 32, 1069 (1983). 3 p. L. Feigner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. R. Northrop, G. M. Ringold, and M. Danielson, Proc. Natl. Acad. Sci. U.S.A. 84, 7413 (1987). 4 R. Malone, P. L. Feigner, and I. Verma, Proc. Natl. Acad. Sci. U.S.A. 86, 6077 (1989). 5 N. DiizgOne~, J. A. Goldstein, D. S. Friend, and P. L. Feigner, Biochemistry 28, 9179 (1989). 6 K. Konopka, L. Stamatatos, C. E. Larsen, B. R. Davis, and N. Dfizgtine~, J. Gen. Virol. 72, 2685 (1991). 7 g. J. Debs, L. P. Freedman, S. Edmunds, K. L. Gaensler, N. Dgzgtine¢, and K. R. Yamamoto, ft. Biol. Chem. 265, 10189 (1990). METHODSIN ENZYMOL(X~Y,VOL. 221
Copyright© 1993by AcademicPress,Inc. Allrightsoftel~rc~uctlonin any formr~crved.
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[23]
CA). To ascertain the complete absence of bacteria or fungi, an aliquot of cells may be incubated periodically in medium without the antibiotics, because undetectable numbers of microorganisms may still grow in the presence of antibiotics. The cells are washed three times with phosphatebuffered saline (PBS). Thirty micrograms of Lipofectin reagent (Life Technologies, Inc., Gaithersburg, MD) in 1.5 ml Opfi-MEM I medium (Life Technologies) is mixed with 5 #g DNA in 1.5 ml Opti-MEM I. Different amounts oflipofectin and DNA may be optimal for different cell lines. The lipofectin reagent and DNA may also be added sequentially (but without washing away) to culture medium consisting of Opti-MEM I. The cells are incubated for 6 hr in a humidified CO2 (5-10%) incubator. Three milliliters of medium containing 20% (v/v) FBS is added to the plates, and the calls are incubated for another 24-48 hr. When preparing complexes of DNA and Lipofectin, it is essential to maintain a net positive charge on the complex. Optimal transfection occurs when the ratio of the molar equivalent positive charge contributed by DOTMA to the molar equivalent negative charge contributed by the nucleic acid is in the range I. 1-2.5. The corresponding ratio of the weight of the lipid (i.e., Lipofectin) to the weight of the nucleic acid is about 4 - 10. The above procedures should be optimized for each cell line used. It is first necessary to determine the toxic levels of Lipofecfin. The Lipofecfion experiments can then be performed at about half the toxic concentration, with varying concentrations of nucleic acid in different culture plates to produce the maximal transfection. When quantifies of nucleic acid (e.g., plasmid) are limited, a reasonable amount of plasmid is added to several culture plates, and the amount of lipofectin may be varied below the toxic range. Lipofectin-mediated transfection has been used to deliver plasmids such as pRSV-CAT, using chloramphenicol acetyltransferase activity as a marker for the delivery of the plasmid,4 and pSV2-LacZ, using fl-galactosidase activity as a marker, s Delivery of Transcription Factors Purified transcription factors can be delivered to cultured cells by forming a complex of the protein, the reporter plasmid containing the response element, and DOTMA liposomes. Although the commercially available lipofectin reagent can be used directly for this application, the use of pure DOTMA (obtained from Syntex Research, Palo Alto, CA) liposomes results in substantially higher levels of delivery.~ Liposomes are s j. Feigner, R. Kumar, R. Border, and P. L. Feigner, Z Cell Biol. 111, 381a (1990).
[23]
DELIVERY BY CATIONIC LIPOSOMES
305
prepared by sonication under an argon atmosphere (produced by purging the glass tube with argon for 15 scc), in a bath-type sonicator (Laboratory Supply Co., Hicksville, NY), following the formation of a dry film of lipid by rotary evaporation. 9 Cells (e.g., CV-1 or HTC) are plated at a density of 106/100-mm diameter plastic Petri dishes in DME H-21 medium containing I0% (v/v) FBS, and incubated overnight in a CO2 incubator at 37 °. The cells are treated for 1 hr with 100 ]LM"chloroquine, washed twice with PBS, and a mixture of liposomes, transcription factor, and reporter plasmid is added to the cells in DMEM without serum, as described below. The optimal concentrations of the transcription factor and plasmid to be used are determined empirically. The transcription factor (20-125 #g) is mixed gently with reporter plasmid (0.5-2.5#g) and DOTMA liposomes (25 nmol) in 1 ml of DMEM. This mixture (l ml) is placed in each petri dish containing the cells, and 4 ml of DMEM with 100 ~ chloroquine and 0.05% (v/v) gentamicin is added immediately afterward. After the cells are incubated for 5 hr at 37 °, l0 ml of DMEM/10% (v/v) b-'BSis added and the cells cultured for another 12 hr. The cells are then washed twice with PBS, and incubated in DMEM/10% (v/v) FBS for 24-36 hr. The transcription factor used in our studies is the glucocorticoid receptor derivative T7X556, the segment of the glucocorticoid receptor from amino acid 407 to 556 (with additional nonreceptor amino acids at the C and N termini added during expression in Escherichia coli), which includes the DNA-binding region but lacks the hormone-binding region.7 The reporter plasmids contain (1) the glucocorticoid response element, (2) a promoter, and (3) the gene encoding chloramphenicol acetyltransferase (CAT). For example, the plasmid GTCO 7 contained a 46-base pair synthetic glucocorticoid response element (GRE) l° fused to the herpes simplex virus thymidine kinase promoter. To determine the level of transcription, the cells are washed twice with PBS, scraped from the plate with a rubber policeman or Teflon cell scraper, centrifuged (1000 g, 5 min, 4°), resuspcnded in a small volume (100-200 #l) of 250 mM Tris (pH 7.5), freeze-thawed three times, incubated at 65 ° for 10 rain, and centrifuged again (12,500 g, 10 rain, 4°). The protein concentration in the supernatant is determined and 20 #g of supernatant protein is placed into a standard CAT assay.n This method has been used to show that the transcriptional regulator T7X556 localizes in the nucleus following intracellular delivery, and that it 9 N. Diizgiine~ and J. Wilschut, this series, Vol. 220 [1]. m D. D. Sakai, S. Helms, J. Carlstcdt-Duke, J. A. Gustafsson, F. M. Rottman, and K. R. Yarnamoto, Genes Dev. 2, 1144 (1988). n C. M. Gorman, L. F. Moffat, and B. H. Howard, Mol. Cell. Biol. 2, 1044 (1982).
306
MACROMOLECULE I N T R O D U C T I O N BY M E M B R A N E F U S I O N
[24]
selectively enhances expression from promoters linked to the glucocorticoid response element.7 These experiments have also demonstrated that the expression of the transcriptional regulator in bacteria and subsequent biochemical purification does not affect the in vivo activities of the molecule, as shown by endogenous expression in mammalian cells.
[24] M i c r o i n j e c t i o n o f M a c r o m o l e c u l e s i n t o C u l t u r e d Cells by Erythrocyte Ghost-Cell Fusion By YOSHIHIRO YONEDA
Introduction Various methods for introducing macromolecules, such as proteins and nucleic acids (DNA and RNA), into cultured cells have been developed and used to analyze the biological activities of these molecules in living cells. One of the most useful and commonly used methods is microinjection with a microcapillary. By this method, foreign substances can be introduced precisely into single cells. However, this method requires a special apparatus and skillful techniques. An alternative method is the widely used erythrocyte ghost-cell fusion method mediated by hemagglutinating virus of Japan (HVJ, or Sendai virus). This method is easier, and has the special advantage that it can be used for many cells at the same time. Furusawa et al. first demonstrated the reliability of erythrocyte ghost-cell fusion with fluorescein isothiocyanate (FITC) as a marker. 1 At first the injection frequency was low, but now it has been increased due to various improvements in the technique. This chapter describes the improved erythrocyte ghost-cell fusion method and its applications in biological studies. Materials
Phosphate-buffered saline [PBS(-)]: 137raM NaC1, 2.7mM KC1, 8.1 mMNa2HPO4, 1.5 mM KH2PO4; pH 7.2 Reverse PBS (rPBS): 137 rnM KCI, 2.7 m M NaCI, 8.1 mM Na2HPO4, 1.5 mM KH2PO4; pH 7.2 BSS(-): 140 m M NaC1, 54 mM KC1, 0.34 mM Na~HPO4, 0.44 mM KH2PO4, 10 mMTris-HC1; pH 7.6 BSS(+): BSS(-) + 2 m M CaC12 M. Furusawa, T. Nishimura, M. Yamaizumi, and Y. Okada, Nature (London) 249, 449 (1974). METHODS 1N ENZYMOL(X}Y, VOL. 221
C.opyright © 1993 b y A ~ c ~ Inc. All rights °frepr°ducli°n in any f°rm rlmm'ved"
DELIVERY BY CATIONIC LIPOSOMES
303
[23] I n t r a c e l l u l a r D e l i v e r y o f N u c l e i c A c i d s a n d Transcription Factors by Cationic Liposomes
By NEJAT D O Z G O N E ~ and PHILIP L. FELGNER Liposomes of various types have been used for the delivery of nucleic acids into cultured cells, t Earlier studies involved the encapsulation of the nucleic acids inside liposomes and the delivery of liposomes most likely via an endocytotic pathway.2 Liposomes containing the cationic lipid N-[1(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA) have been found to mediate the efficient delivery of DNA 3 and RNA4 into cells by forming a positively charged complex with the nucleic acids. Although the mechanism of delivery is not well understood, DOTMA-containing liposomes have been shown to undergo fusion with negatively charged liposomess and cells.3,6 DOTMA liposomes have also been used to deliver purified transcription factors into cells, mediating the expression of specific genes. ~ Procedures for transfection and delivery of regulatory proteins are described below. Delivery of Nucleic Acids Cells to be transfectedare plated on 60-ram diameter plastictissue culture platesat a celldensity of 0.5 × 10~/platc,and incubated overnight to become approximately 80% confluent.The transfcctionprocedure may cause toxicityin some celllinesif performed at lower celldensities.The culture medium is composed of Eagle's or Dulbccco's minimal essential medium ( D M E M ) with 10% (v/v) fctal bovine serum and Fungi-Bact solution (penicillin,streptomycin,and Fungizone; IrvineScientific,Irvine, i R. M. Straubinger and D. Papahadjopoulos, this series, Vol. 101, p. 512. : R. M. Stmubinger, K. Hong, D. S. Friend, and D. Papahadjopoulos, Cell (Cambridge, Mass.) 32, 1069 (1983). 3 p. L. Feigner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. R. Northrop, G. M. Ringold, and M. Danielson, Proc. Natl. Acad. Sci. U.S.A. 84, 7413 (1987). 4 R. Malone, P. L. Feigner, and I. Verma, Proc. Natl. Acad. Sci. U.S.A. 86, 6077 (1989). 5 N. DiizgOne~, J. A. Goldstein, D. S. Friend, and P. L. Feigner, Biochemistry 28, 9179 (1989). 6 K. Konopka, L. Stamatatos, C. E. Larsen, B. R. Davis, and N. Dfizgtine~, J. Gen. Virol. 72, 2685 (1991). 7 g. J. Debs, L. P. Freedman, S. Edmunds, K. L. Gaensler, N. Dgzgtine¢, and K. R. Yamamoto, ft. Biol. Chem. 265, 10189 (1990). METHODSIN ENZYMOL(X~Y,VOL. 221
Copyright© 1993by AcademicPress,Inc. Allrightsoftel~rc~uctlonin any formr~crved.
304
MACROMOLECULE INTRODUCTION BY MEMBRANE FUSION
[23]
CA). To ascertain the complete absence of bacteria or fungi, an aliquot of cells may be incubated periodically in medium without the antibiotics, because undetectable numbers of microorganisms may still grow in the presence of antibiotics. The cells are washed three times with phosphatebuffered saline (PBS). Thirty micrograms of Lipofectin reagent (Life Technologies, Inc., Gaithersburg, MD) in 1.5 ml Opfi-MEM I medium (Life Technologies) is mixed with 5 #g DNA in 1.5 ml Opti-MEM I. Different amounts oflipofectin and DNA may be optimal for different cell lines. The lipofectin reagent and DNA may also be added sequentially (but without washing away) to culture medium consisting of Opti-MEM I. The cells are incubated for 6 hr in a humidified CO2 (5-10%) incubator. Three milliliters of medium containing 20% (v/v) FBS is added to the plates, and the calls are incubated for another 24-48 hr. When preparing complexes of DNA and Lipofectin, it is essential to maintain a net positive charge on the complex. Optimal transfection occurs when the ratio of the molar equivalent positive charge contributed by DOTMA to the molar equivalent negative charge contributed by the nucleic acid is in the range I. 1-2.5. The corresponding ratio of the weight of the lipid (i.e., Lipofectin) to the weight of the nucleic acid is about 4 - 10. The above procedures should be optimized for each cell line used. It is first necessary to determine the toxic levels of Lipofecfin. The Lipofecfion experiments can then be performed at about half the toxic concentration, with varying concentrations of nucleic acid in different culture plates to produce the maximal transfection. When quantifies of nucleic acid (e.g., plasmid) are limited, a reasonable amount of plasmid is added to several culture plates, and the amount of lipofectin may be varied below the toxic range. Lipofectin-mediated transfection has been used to deliver plasmids such as pRSV-CAT, using chloramphenicol acetyltransferase activity as a marker for the delivery of the plasmid,4 and pSV2-LacZ, using fl-galactosidase activity as a marker, s Delivery of Transcription Factors Purified transcription factors can be delivered to cultured cells by forming a complex of the protein, the reporter plasmid containing the response element, and DOTMA liposomes. Although the commercially available lipofectin reagent can be used directly for this application, the use of pure DOTMA (obtained from Syntex Research, Palo Alto, CA) liposomes results in substantially higher levels of delivery.~ Liposomes are s j. Feigner, R. Kumar, R. Border, and P. L. Feigner, Z Cell Biol. 111, 381a (1990).
[23]
DELIVERY BY CATIONIC LIPOSOMES
305
prepared by sonication under an argon atmosphere (produced by purging the glass tube with argon for 15 scc), in a bath-type sonicator (Laboratory Supply Co., Hicksville, NY), following the formation of a dry film of lipid by rotary evaporation. 9 Cells (e.g., CV-1 or HTC) are plated at a density of 106/100-mm diameter plastic Petri dishes in DME H-21 medium containing I0% (v/v) FBS, and incubated overnight in a CO2 incubator at 37 °. The cells are treated for 1 hr with 100 ]LM"chloroquine, washed twice with PBS, and a mixture of liposomes, transcription factor, and reporter plasmid is added to the cells in DMEM without serum, as described below. The optimal concentrations of the transcription factor and plasmid to be used are determined empirically. The transcription factor (20-125 #g) is mixed gently with reporter plasmid (0.5-2.5#g) and DOTMA liposomes (25 nmol) in 1 ml of DMEM. This mixture (l ml) is placed in each petri dish containing the cells, and 4 ml of DMEM with 100 ~ chloroquine and 0.05% (v/v) gentamicin is added immediately afterward. After the cells are incubated for 5 hr at 37 °, l0 ml of DMEM/10% (v/v) b-'BSis added and the cells cultured for another 12 hr. The cells are then washed twice with PBS, and incubated in DMEM/10% (v/v) FBS for 24-36 hr. The transcription factor used in our studies is the glucocorticoid receptor derivative T7X556, the segment of the glucocorticoid receptor from amino acid 407 to 556 (with additional nonreceptor amino acids at the C and N termini added during expression in Escherichia coli), which includes the DNA-binding region but lacks the hormone-binding region.7 The reporter plasmids contain (1) the glucocorticoid response element, (2) a promoter, and (3) the gene encoding chloramphenicol acetyltransferase (CAT). For example, the plasmid GTCO 7 contained a 46-base pair synthetic glucocorticoid response element (GRE) l° fused to the herpes simplex virus thymidine kinase promoter. To determine the level of transcription, the cells are washed twice with PBS, scraped from the plate with a rubber policeman or Teflon cell scraper, centrifuged (1000 g, 5 min, 4°), resuspcnded in a small volume (100-200 #l) of 250 mM Tris (pH 7.5), freeze-thawed three times, incubated at 65 ° for 10 rain, and centrifuged again (12,500 g, 10 rain, 4°). The protein concentration in the supernatant is determined and 20 #g of supernatant protein is placed into a standard CAT assay.n This method has been used to show that the transcriptional regulator T7X556 localizes in the nucleus following intracellular delivery, and that it 9 N. Diizgiine~ and J. Wilschut, this series, Vol. 220 [1]. m D. D. Sakai, S. Helms, J. Carlstcdt-Duke, J. A. Gustafsson, F. M. Rottman, and K. R. Yarnamoto, Genes Dev. 2, 1144 (1988). n C. M. Gorman, L. F. Moffat, and B. H. Howard, Mol. Cell. Biol. 2, 1044 (1982).
306
MACROMOLECULE I N T R O D U C T I O N BY M E M B R A N E F U S I O N
[24]
selectively enhances expression from promoters linked to the glucocorticoid response element.7 These experiments have also demonstrated that the expression of the transcriptional regulator in bacteria and subsequent biochemical purification does not affect the in vivo activities of the molecule, as shown by endogenous expression in mammalian cells.
[24] M i c r o i n j e c t i o n o f M a c r o m o l e c u l e s i n t o C u l t u r e d Cells by Erythrocyte Ghost-Cell Fusion By YOSHIHIRO YONEDA
Introduction Various methods for introducing macromolecules, such as proteins and nucleic acids (DNA and RNA), into cultured cells have been developed and used to analyze the biological activities of these molecules in living cells. One of the most useful and commonly used methods is microinjection with a microcapillary. By this method, foreign substances can be introduced precisely into single cells. However, this method requires a special apparatus and skillful techniques. An alternative method is the widely used erythrocyte ghost-cell fusion method mediated by hemagglutinating virus of Japan (HVJ, or Sendai virus). This method is easier, and has the special advantage that it can be used for many cells at the same time. Furusawa et al. first demonstrated the reliability of erythrocyte ghost-cell fusion with fluorescein isothiocyanate (FITC) as a marker. 1 At first the injection frequency was low, but now it has been increased due to various improvements in the technique. This chapter describes the improved erythrocyte ghost-cell fusion method and its applications in biological studies. Materials
Phosphate-buffered saline [PBS(-)]: 137raM NaC1, 2.7mM KC1, 8.1 mMNa2HPO4, 1.5 mM KH2PO4; pH 7.2 Reverse PBS (rPBS): 137 rnM KCI, 2.7 m M NaCI, 8.1 mM Na2HPO4, 1.5 mM KH2PO4; pH 7.2 BSS(-): 140 m M NaC1, 54 mM KC1, 0.34 mM Na~HPO4, 0.44 mM KH2PO4, 10 mMTris-HC1; pH 7.6 BSS(+): BSS(-) + 2 m M CaC12 M. Furusawa, T. Nishimura, M. Yamaizumi, and Y. Okada, Nature (London) 249, 449 (1974). METHODS 1N ENZYMOL(X}Y, VOL. 221
C.opyright © 1993 b y A ~ c ~ Inc. All rights °frepr°ducli°n in any f°rm rlmm'ved"