l. 1992 Oxford University Press
Nucleic Acids Research, 1992, Vol. 20, No. 23 6425-6426
High-efficiency gene transfer into cardiac myocytes Hao Xu, Jean Miller and Bruce T.Liang* Department of Medicine, Cardiovascular Division, and Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Received July 27, 1992; Revised and Accepted October 6, 1992 Transfection of DNA into cultured cardiac myocytes provides a unique opportunity to study gene expression and structure -function relationship of myocardial proteins. Because of the lack of cell line, transfection of cardiac myocytes has been limited to primary cultures of heart cells. Prior study demonstrated a very low efficiency ( < 1 %) of transfection of the foreign genes into the primary cultures of embryonic chick heart cells (1). A substantial increase in the transfection efficiency will facilitate quantitative determination of the expression of foreign genes and of the effects of such expression on the function and behavior of cardiac cells. In the present study, we used the same primary cultures of embryonic chick heart cells to test the ability of a new modification of the calcium phosphate precipitate method of transfection. A trial and ventricular myocytes from chick embryos 14 days in ovo were maintained in cultures for 24 hours as previously described (2, 3). The cultured cells were then exposed to either calcium phosphate alone (Control) or calcium phosphate plus a plasmid containing a functional Lac Z gene that was driven from the SV40 Promoter, PSV-,B-gal (4). The concentrations of the various components in the calcium phosphate/plasmid cocktail were listed in Table 1. The principal feature of the current method, as different from the previously reported calcium phosphate procipitate-mediated transfection, was to ensure the formation of coarse precipitates of calcium phosphate/DNA mixture. This was accomplished by mixing the DNA with CaC12 first and followed by addition of a Hepesbuffered solution containing the following (mM): KCl (10), D(+) glucose (11), Na2HPO4 (2.09-2.79), Hepes (42), NaCl (171). The concentration of Na2HPO4 was varied as a function of the amount of plasmid in the cocktail. The cocktail was added dropwise to the culture media (M/199 media supplemented by 6% fetal bovine serum) directly with simultaneous swirling of the media. The cultures were then returned to a CO2 incubator in which the temperature was set at 37°C and CO2 at 4% for an additional six hours. The presence of coase precipitates was determined by microscopic examination of the culture media and was directly correlated with high-efficiency transfection subsequently. Decreasing the concentration of Na2HPO4 to 0.07 mM resulted in the appearance of fine precipitates that was correlated with a decreased % transformants (data submitted but not shown). The media containing the precipitates were removed and fresh media added, followed by further incubation of the cultures for 24-48 hours; at which times, cells that were positive for 3-galactosidase (,3-gal) were identified with the chromogenic substrate X-gal (5-bromo-4-chloro-indolyl-,B-galactopyranoside)
a
b
Figure 1. Cultured ventricular cells were grown to 50-70% confluency, transfected with (a) calcium phosphate alone or with (b) calcium phosphate plus pSV-,B-gal. Prior to staining with X-gal, the cultures were treated with trypsin/EDTA (Sigma Cell Culture Reagents) to detach the cells and trypsin neutralized with a solution of donor horse serum and Hanks' Balanced Salt Solution (50/50, v/v). The isolated cells were collected and resuspended in the X-gal solution directly for identification of the blue-stained cells. The X-gal solution contains the followings: 1 mM MgCl2, 3.3 mM K4Fe(CN)6,3H20, 3.3 mM K3Fe(CN)6, 150 mM NaCI, 10 mM sodium phosphate buffer (pH = 7.0) and 0.2% X-gal.
* To whom correspondence should be addressed at: 504 Johnson Pavilion, 36th Street and Hamilton Walk, PA 19104, USA
University of Pennsylvania, School of Medicine, Philadelphia,
6426 Nucleic Acids Research, 1992, Vol. 20, No. 23 Table 1. Components
Final concentration (mM)
KCL CaCl2 (+) Glucose Na2HPO4 NaCl Hepes
0.5 12.5 0.55 0.1-0.13 8.55 2. 1
The concentrations represent those present in the culture media. The concentration of Na2HPO4 was varied according to the plasmid concentration (0-20 yg: 0.1 mM; > 20 ug: 0.13 mM).
(5). The identification of X-gal-positive cells were carried out in both cell suspensions and monolayer on the culture plate. The data demonstrate the following: 1. Cultured cells exposed to calcium phosphate or calcium phosphate/plasmid mixture formed a monolayer that exhibited similarly strong spontaneous contraction. 2. None of the cultures exposed to calcium phosphate alone showed any ,3-gal-positive cells (n = 6 different cultures). 3. Both ventricular and atrial (data not shown) cultures can be efficiently transfected with this method. Parallel staining of the myocytes with periodic acid Schiff method (6) (>90% of the cultured cells were myocytes and as per reference 7) and X-gal staining of the 13-gal-positive cells demonstrate that majority of the 13-gal-positive cells are myocytes 40% transformants, > 10 Rg (usually 15-20 Ag) of the plasmid DNA was required. Awaiting 48 hours after the exposure to calcium phosphate/plasmid did not change the level of expression of the Lac Z gene in either the atrial or the ventricular cultures. Taken together, the current modified method of calcium phosphate-mediated gene transfer represents a significant improvement in the efficiency of transfection into cultured cardiac myocytes and should be useful in studies of cardiac gene expression.
ACKNOWLEDGEMENT The authors would like to thank Dr Judith L.Swain for useful discussion and pithy comments.
REFERENCES 1. 2. 3. 4.
Antin,P.B., Mar,J.H. and Ordahl,C.P. (1988) Biotechniques 6, 640-649. Liang,B.T. and Galper,J.B. (1988) Biochem. Pharmacol. 37, 4549-4555. Liang,B.T. (1989) J. Pharmacol. Exp. Ther. 249, 775-784. Edlund,T,, Walker,M.D., Barr,P.J. and Rutter,W.J. (1985) Science 230, 912-916. 5. Fischer,J.A., Giniger,E., Maniatis,T. and Ptashne,M. (1989) Nature 332, 853-856. 6. Lillie,R.D. and Fullmer,H.M. (1976) Histopathologic Technique and Practical Histochemistry. McGraw-Hill, New York. 7. Marsh,J.D., Lachance,D. and Kim,D. (1985) Circ. Res. 57, 171-181.
Nucleic Acids Research, 1992, Vol. 20, No. 23 6425-6426
High-efficiency gene transfer into cardiac myocytes Hao Xu, Jean Miller and Bruce T.Liang* Department of Medicine, Cardiovascular Division, and Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Received July 27, 1992; Revised and Accepted October 6, 1992 Transfection of DNA into cultured cardiac myocytes provides a unique opportunity to study gene expression and structure -function relationship of myocardial proteins. Because of the lack of cell line, transfection of cardiac myocytes has been limited to primary cultures of heart cells. Prior study demonstrated a very low efficiency ( < 1 %) of transfection of the foreign genes into the primary cultures of embryonic chick heart cells (1). A substantial increase in the transfection efficiency will facilitate quantitative determination of the expression of foreign genes and of the effects of such expression on the function and behavior of cardiac cells. In the present study, we used the same primary cultures of embryonic chick heart cells to test the ability of a new modification of the calcium phosphate precipitate method of transfection. A trial and ventricular myocytes from chick embryos 14 days in ovo were maintained in cultures for 24 hours as previously described (2, 3). The cultured cells were then exposed to either calcium phosphate alone (Control) or calcium phosphate plus a plasmid containing a functional Lac Z gene that was driven from the SV40 Promoter, PSV-,B-gal (4). The concentrations of the various components in the calcium phosphate/plasmid cocktail were listed in Table 1. The principal feature of the current method, as different from the previously reported calcium phosphate procipitate-mediated transfection, was to ensure the formation of coarse precipitates of calcium phosphate/DNA mixture. This was accomplished by mixing the DNA with CaC12 first and followed by addition of a Hepesbuffered solution containing the following (mM): KCl (10), D(+) glucose (11), Na2HPO4 (2.09-2.79), Hepes (42), NaCl (171). The concentration of Na2HPO4 was varied as a function of the amount of plasmid in the cocktail. The cocktail was added dropwise to the culture media (M/199 media supplemented by 6% fetal bovine serum) directly with simultaneous swirling of the media. The cultures were then returned to a CO2 incubator in which the temperature was set at 37°C and CO2 at 4% for an additional six hours. The presence of coase precipitates was determined by microscopic examination of the culture media and was directly correlated with high-efficiency transfection subsequently. Decreasing the concentration of Na2HPO4 to 0.07 mM resulted in the appearance of fine precipitates that was correlated with a decreased % transformants (data submitted but not shown). The media containing the precipitates were removed and fresh media added, followed by further incubation of the cultures for 24-48 hours; at which times, cells that were positive for 3-galactosidase (,3-gal) were identified with the chromogenic substrate X-gal (5-bromo-4-chloro-indolyl-,B-galactopyranoside)
a
b
Figure 1. Cultured ventricular cells were grown to 50-70% confluency, transfected with (a) calcium phosphate alone or with (b) calcium phosphate plus pSV-,B-gal. Prior to staining with X-gal, the cultures were treated with trypsin/EDTA (Sigma Cell Culture Reagents) to detach the cells and trypsin neutralized with a solution of donor horse serum and Hanks' Balanced Salt Solution (50/50, v/v). The isolated cells were collected and resuspended in the X-gal solution directly for identification of the blue-stained cells. The X-gal solution contains the followings: 1 mM MgCl2, 3.3 mM K4Fe(CN)6,3H20, 3.3 mM K3Fe(CN)6, 150 mM NaCI, 10 mM sodium phosphate buffer (pH = 7.0) and 0.2% X-gal.
* To whom correspondence should be addressed at: 504 Johnson Pavilion, 36th Street and Hamilton Walk, PA 19104, USA
University of Pennsylvania, School of Medicine, Philadelphia,
6426 Nucleic Acids Research, 1992, Vol. 20, No. 23 Table 1. Components
Final concentration (mM)
KCL CaCl2 (+) Glucose Na2HPO4 NaCl Hepes
0.5 12.5 0.55 0.1-0.13 8.55 2. 1
The concentrations represent those present in the culture media. The concentration of Na2HPO4 was varied according to the plasmid concentration (0-20 yg: 0.1 mM; > 20 ug: 0.13 mM).
(5). The identification of X-gal-positive cells were carried out in both cell suspensions and monolayer on the culture plate. The data demonstrate the following: 1. Cultured cells exposed to calcium phosphate or calcium phosphate/plasmid mixture formed a monolayer that exhibited similarly strong spontaneous contraction. 2. None of the cultures exposed to calcium phosphate alone showed any ,3-gal-positive cells (n = 6 different cultures). 3. Both ventricular and atrial (data not shown) cultures can be efficiently transfected with this method. Parallel staining of the myocytes with periodic acid Schiff method (6) (>90% of the cultured cells were myocytes and as per reference 7) and X-gal staining of the 13-gal-positive cells demonstrate that majority of the 13-gal-positive cells are myocytes 40% transformants, > 10 Rg (usually 15-20 Ag) of the plasmid DNA was required. Awaiting 48 hours after the exposure to calcium phosphate/plasmid did not change the level of expression of the Lac Z gene in either the atrial or the ventricular cultures. Taken together, the current modified method of calcium phosphate-mediated gene transfer represents a significant improvement in the efficiency of transfection into cultured cardiac myocytes and should be useful in studies of cardiac gene expression.
ACKNOWLEDGEMENT The authors would like to thank Dr Judith L.Swain for useful discussion and pithy comments.
REFERENCES 1. 2. 3. 4.
Antin,P.B., Mar,J.H. and Ordahl,C.P. (1988) Biotechniques 6, 640-649. Liang,B.T. and Galper,J.B. (1988) Biochem. Pharmacol. 37, 4549-4555. Liang,B.T. (1989) J. Pharmacol. Exp. Ther. 249, 775-784. Edlund,T,, Walker,M.D., Barr,P.J. and Rutter,W.J. (1985) Science 230, 912-916. 5. Fischer,J.A., Giniger,E., Maniatis,T. and Ptashne,M. (1989) Nature 332, 853-856. 6. Lillie,R.D. and Fullmer,H.M. (1976) Histopathologic Technique and Practical Histochemistry. McGraw-Hill, New York. 7. Marsh,J.D., Lachance,D. and Kim,D. (1985) Circ. Res. 57, 171-181.
