- Email: [email protected]
Direct Gene Transfer into the Mouse Heart
J Mol Cell Cardiol 29, 1499–1504 (1997)
Direct Gene Transfer into the Mouse Heart Kai Li, Robert E. Welikson, Karen L. Vikstrom and Leslie A. Leinwand Department of Microbiology and Immunology, Albert Einstein College of Medicine, NY, and the Department of Molecular Cellular Developmental Biology, University of Colorado, CO, USA (Received 30 May 1996, accepted in revised form 21 January 1997) K. L, R. E. W, K. L. V L. A. L. Direct Gene Transfer into the Mouse Heart. Journal of Molecular and Cellular Cardiology (1997) 29, 1499–1504. Direct injection of plasmid DNA into the myocardium of several species has been shown to be useful for studying cardiac gene expression. However, despite a better understanding of mouse genetics and the availability of several disease models in mice, gene injection with plasmid DNA into the mouse heart has not been reported. In this study, we demonstrate a simple and reproducible method for gene transfer into the mouse heart via direct injection of plasmid DNA. A firefly luciferase gene, driven by the RSV promoter, was used to quantitatively determine the spatial and temporal characteristics of gene transfer. Luciferase gene expression was stable for 8 weeks and showed a dose-dependent response over a range of 0.3–3 lg of input DNA. Inter-animal variability was low and gene expression was restricted to the left ventricle, near the site of injection. This method was also demonstrated to be suitable for detecting the expression of structural genes under the control of cellular promoters. Immunohistochemistry was used to detect the expression of an epitope-tagged myosin heavy chain driven by a rat a-myosin heavy chain promoter. Thus, naked DNA injection into the mouse heart results in a highly reproducible expression of constructs with either viral or cellular promoters. It is a relatively inexpensive and efficient means of studying cardiac gene regulation in vivo and a useful tool for screening the potential transgenes before generating transgenic mice. 1997 Academic Press Limited
K W: Gene expression; Mouse heart; Gene injection.
Introduction Since the initial demonstration of direct gene injection into mouse skeletal muscle (Wolff et al., 1990), gene injection into various skeletal muscle sites has been studied, and various parameters for optimising gene transfer have been tested. A mouse’s hind limbs, diaphragm, and tongue can each take up and express injected genes (Acsadi et al., 1991a; Wolff et al., 1991; 1992; Davis et al., 1993). Gene transfer into a normal adult skeletal muscle of mouse and rat is quite inefficient, although agents which promote muscle degeneration/regeneration dramatically increase the expression of injected genes (Davis and Jasmin, 1993; Danko et al., 1994). In contrast to skeletal muscle, gene injection into the rat heart is quite
efficient, and results in up to 50-fold higher levels of gene expression than similar experiments in skeletal muscles (Lin et al., 1990; Kitsis et al., 1991; Buttrick et al., 1992). Transfer of naked plasmid DNA into the heart has subsequently been demonstrated in rabbits and dogs (Gal et al., 1993; von Harsdorf et al., 1993). Although the mechanism underlying the uptake of injected genes remains unknown, it appears that this ability is restricted to striated muscles. In rat and rabbit hearts, gene expression after a single DNA injection is generally stable over time. The potential applications of direct gene transfer include potential somatic gene therapy, the analysis of gene regulation in vivo (Kitsis et al., 1991; Buttrick et al., 1992), vaccination (Raz et al., 1994), and hormone replacement therapy (Leinwand and Leiden, 1991).
Please address all correspondence to: Leslie A. Leinwand, Department of Molecular Cellular Developmental Biology, Campus Box 347, University of Colorado, Boulder, CO 80309-0347, USA.
0022–2828/97/051499+06 $25.00/0
mc970389
1997 Academic Press Limited
1500
K. Li et al.
Despite the prevalent use of the mouse in genetic and developmental studies, there are no published reports of DNA injections into the mouse heart. We have developed surgical protocols for mouse cardiac injection and characterized the resulting gene expression. Our data show that gene injection into the mouse heart is highly reproducible, and results in stable gene expression over 8 weeks. When reporter genes were used, expression was dosedependent and restricted to a limited area of the left ventricle. These results are consistent with previous reports of gene injection in rat (Buttrick et al., 1992) and dog hearts (von Harsdorf et al., 1993). The detection of a tagged mammalian contractile protein gene under the control of a cellular promoter, suggests that it may also be possible to evaluate candidate transgenes with this method. The consistency of the results obtained with gene injection into the mouse heart has opened a new avenue for studying mouse cardiac gene expression in vivo, using the most prevalent species in mammalian genetic analysis.
Materials and Methods Adult female CD1 mice were anesthetized with chloral hydrate (0.4 g/kg, i.p.). The animals were placed in a supine position and the upper limbs were taped to a table. Chest skin was cleaned with 70% ethanol and a 1–1.5 cm incision was made along the left side of the sternum. The muscle layers of the chest wall were bluntly dissected to avoid bleeding. The thorax was opened by cutting the rib at the point of the most pronounced cardiac pulsation. While forceps were used to widen the chest, the abdomen and the right side of the chest were pressed to push the heart out of the thoracic cavity. Unless specified, 5 lg of DNA in 15 ll normal saline was injected through a 30-gauge needle into the left ventricular free wall. Following the injection, the heart was placed back into the thoracic cavity and the chest held closed with forceps. After the muscle layers were closed with one suture, the forceps were released. The chest cavity was gently squeezed to expel air out before tightly tying the suture and stapling the skin. Spontaneous respiration was maintained and no mechanical ventilation was needed. Two plasmids were used for this study. The reporter gene, pRSVluc (Buttrick et al., 1992) contains a firefly luciferase cDNA fused to the long terminal repeat of the Rous Sarcoma Virus (RSV LTR) (Gorman et al., 1982). A candidate transgene paa-myc, encoding a myc epitope-tagged rat a-myosin heavy
chain (aMyh) driven by a 2.9 kb rat aMyh promoter (Vikstrom and Leinwand, unpubl) was also used. For luciferase assays, mice were killed by cervical dislocation 1, 2, 4, and 8 weeks after gene injection. The hearts were removed and washed with icecold homogenization buffer (25 m Gly-gly, 15 m MgSO4, 4 m EGTA, and 4 m DTT). The tissue was minced in 0.4 ml homogenization buffer, homogenized for 20 s with a Tissuemizer (Tekmar Company, Cincinnati, OH, USA), and then centrifuged at 5000×g for 20 min (4°C). The supernatant was removed and kept for luciferase assays. Twenty-five percent of the supernatant (100 ll) was mixed with 360 ll reaction buffer (25 m Gly-gly, 15 m MgSO4, 4 m EGTA, 4 m DTT, 15 m KPO4, pH 7.8, and 2 m Na+-ATP). The enzymatic reaction was initiated by injection of 100 ll homogenization buffer containing 0.2 m D-luciferin and light emission was measured for 20 sec (Brasier et al., 1989). The light units measured from the 100 ll of supernatant were expressed as relative luciferase activity. For tissue sectioning and immunostaining, the heart was harvested 5 days after gene injection, flash frozen in liquid nitrogen and stored at −80°C. After embedding in OCT, 5 lm sections were collected every 20 sections and placed on poly--lysine coated slides. The slides were airdried, fixed in acetone (−20°C for 5 min), and then allowed to air dry again. Endogenous peroxidase activity was blocked by incubating in 0.3% hydrogen peroxide in methanol for 30 min at room temperature. All subsequent incubations were done at 37°C in a humidified chamber. After air drying, the slides were blocked by incubating for 30 min with 10% goat serum in phosphate buffered saline (PBS). The sections were then incubated with 9E10.2 culture supernatant (Bishop and Evan, 1985; ATCC CRL 1729) for 30 min. The slides were washed by soaking in three, 5 min changes of PBS and then incubated for 30 min with a 1:200 dilution (in 10% goat serum) of horseradish peroxidase conjugated goat anti-mouse IgG (Bio-Rad, Hercules, CA, USA). Slides were washed as above, and color was developed using the DAB substrate kit (Vector Laboratories, Inc., California, CA, USA), according to the manufacturer’s instructions. Slides were mounted with Airvol (Air Products & Chemical Inc., Allentown, PA, USA). All results are expressed as mean±... A oneway analysis of variance followed by a Student– Newman–Keuls test was used for multiple comparisons. A P value less than 0.05 was used as statistical significance.
1501
Direct Gene Transfer into the Mouse Heart 7
6
*
4
2
0
Relative LUC activity (log)
Relative LUC activity (log)
8
10 3 0.3 1 Dosage of DNA injected ( g)
Figure 1 Relative luciferase (LUC) activity measured in mouse myocardial extracts as a function of DNA dose injected. All values of relative luciferase activity were obtained from 100 ll supernatant. Values are mean±... ∗=P<0.05 as compared to the next group with smaller dose.
6
*
5 4 3 2 1 0
4 6 2 Time after injection (weeks)
8
Figure 2 The level of luciferase (LUC) gene expression as a function of time, where n=6 for each group, except n=7 for the 8 week group. Gene expression was stable the 8 weeks examined. ∗=P<0.05 as compared to the other groups.
Results The survival of mice from the surgery and gene injection was quite high (76%, n=55). After gene injection, death resulting from depressed respiration occurred primarily within the first h. To maximise simplicity, no respirator was used for this surgery. With the luciferase reporter gene, high levels of luciferase activity were detected in all surviving mice using doses of DNA from 0.3 to 10 lg. With the exception of one heart, which probably represents a poor gene injection, the inter-animal variability in luciferase activity was less than one order of magnitude within any group. To determine the optimal DNA dose for gene injection into the mouse heart for the RSV-luciferase construct, four doses of pRSVLuc were tested: 0.3, 1, 3, and 10 lg, all in a volume of 15 ll. Each dose of DNA was injected into five mice, and luciferase activity was assayed 1 week later. As shown in Figure 1, luciferase activity increased in a dose-responsive manner with injections of up to 3 lg DNA, then plateaued. This suggests that to achieve gene expression within the linear range of a RSV LTR driven construct, 3 lg of DNA is the optimal dose. By measuring luciferase levels at various times post-injection, we addressed whether the expression of a reporter gene was stable over time (Fig. 2). The highest level of reporter gene expression was detected 1 week after gene injection (P<0.05 as compared to the other three groups). The expression level decreased slightly thereafter, but remained stable until 8 weeks post injection. The spatial distribution of gene expression in
Relative LUC activity (log)
7 6 5 4 3 2 1 0
LV A + RV Septum Space distribution of reporter gene
Figure 3 Spatial distribution of luciferase (LUC) expression after a single injection into the mouse heart. Three hearts were included in this group and all showed high levels of expression in the left ventricle (LV) and no detectable luciferase activity in atrium (A), right ventricle (RV), and septum.
injected mouse hearts was assessed using the luciferase reporter gene. Luciferase assays were performed on dissected regions of the heart (n=3). As shown in Figure 3, only the left ventricle expressed the reporter gene. Since reporter gene assays, such as luciferase activity, are extremely sensitive, we wished to assess whether expression of genes driven by cellular promoters can be detected following gene injection into the mouse heart. Figure 4 shows the distribution of myc-tagged aMyh protein 1 week after injection of an aMyh-promoter-driven construct into the mouse heart. Immunostaining with a myc-specific monoclonal antibody only revealed gene expression around the needle track in the left ventricle (Fig. 4A). Many cardiac myocytes were positive for the myc-tagged Myh protein in this region. However, there were negative cells within
1502
K. Li et al.
Figure 4 Immunostaining for myc tagged rat a myosin heavy chain expression in the mouse heart 1 week after a 5 lg DNA injection of paamyc plasmid. Dark brown staining indicates myc tagged rat a myosin. An injected heart is shown in (A) and an uninjected is shown in (B). Bar equals 20 lm.
the needle track as well. The highest density of positive cells was seen in the central part of the needle track with fewer positive cells around the periphery of the track. There were no positive cells with the myc antibody in an uninjected mouse heart (Fig. 4B).
Discussion Three observations result from this study. Firstly, we have demonstrated the feasibility of direct gene transfer into the mouse heart. Secondly, we have defined the behavior of genes injected into the mouse heart with respect to the spatial and temporal distribution of gene expression, and thus, provided a range of doses at which a high level expression of injected genes can be obtained. The third contribution of this study, is in demonstrating the ability to evaluate potential transgenes in vivo before producing a transgenic line. This finding is notable, given the inefficiency of transfection into cultured cardiac myocytes. Gene injection into the mouse
heart, thus, provides an easy and useful way for direct gene transfer into a widely used animal species. Gene injection into the mouse heart is fast and simple. Neither extensive sterile technique nor artificial respiration is required. A 77% survival rate and a 100% incidence of positive expression demonstrate its reliability and reproducibility. Any variability of gene expression in the hearts of small mammals, such as the rat and the mouse, is probably due to the quality of the injection. The small size and thin free wall of the mouse heart may significantly contribute to the variability in this animal. Our previous gene injection studies in the rat heart included a vital dye in the DNA solution to guide the injection (Buttrick et al., 1992). In this study, no dye was used since a limited injection volume was allowed. However, we observed evident subepicardial edema immediately following the DNA injection which caused the tissue to change color from red to white. If bleeding was avoided and the chest cavity was not open for longer than 2 min, the survival rate was quite high. From several standpoints, the behavior of genes injected into the mouse heart is similar to that observed in the hearts of rats and dogs (Lin et al., 1990; Acsadi et al., 1991a; Buttrick et al., 1992; von Harsdorf et al., 1993). Our luciferase data indicate that only the left ventricle expressed the injected reporter gene, and the results from paamyc injection further showed that expression of the injected gene was largely restricted to the injection site vicinity. Expression from the RSV-LTR driven construct behaved in a dose-dependent manner in the range of 0.3–3 lg DNA. However, given the quantitatively different promoter activities existing in mammalian cells, it is important to define the optimal dosage for each expression construct injected. Gene expression after plasmid DNA injection is generally stable over time, which is one of the advantages of this method when compared to the more transient gene expression following recombinant adenovirus injection (for review see Leinwand and Leiden, 1991; Nadal-Ginard and Mahdavi, 1993). Finding peak gene expression level 1 week post-injection in the mouse heart is consistent with the findings in rats and dogs (Lin et al., 1990; Kitsis et al., 1991; Buttrick et al., 1992; von Harsdorf et al., 1993). The duration of gene expression in the mouse heart is stable over 8 weeks. The result is similar to findings from previous studies in the rat, showing gene expression for up to 2 months following a single injection (Lin et al., 1990; Kitsis et al., 1991; Buttrick et al., 1992). In
Direct Gene Transfer into the Mouse Heart
addition, very stable gene expression, even over 1 year, in mouse skeletal muscles has been demonstrated after a single plasmid DNA injection (Wolff et al., 1992). Whether gene expression in the mouse heart after DNA injection can persist for the same time period is not addressed in this study, but the relatively stable gene expression we observed over 8 weeks suggests that it may be possible. Compared to other species, direct DNA injection into the mouse heart has several obvious advantages for molecular genetic studies. First, the genetics of the mouse are well understood, and genetic manipulations in the mouse are now performed relatively routinely. A major contribution of DNA injection into animal hearts is in the in vivo analysis of gene regulation. An increasing number of cis- and trans-activating factors have been identified for mammalian genes, generally using transfection of cultured cells (Morkin, 1993). Such approaches for studying cardiac gene regulation have been limited by a lack of permanent cardiac cell lines and an inability to transfect adult primary cardiac myocytes in culture. DNA injection into the mouse heart should become very useful for the study of cardiac gene regulation in vivo, as it has been in the rat (Kitsis et al., 1991; Buttrick et al., 1992). Second, many mouse models for human diseases can be substrates for testing gene therapeutic interventions. One example is the mdx mouse, which is deficient in dystrophin, and has been used to test several approaches in the treatment of muscular dystrophies. These approaches include gene transfer by either direct DNA injection (Acsadi et al., 1991b) or viral vector infection (Karpati and Acsadi, 1994). In addition, following direct DNA injection of a tRNA suppressor gene into the mdx mouse heart, we recently observed a phenotypic correction of cardiac myocytes (Li and Leinwand, in preparation). Since the myocardium takes up DNA more efficiently than other tissue types, gene injection into the mouse heart provides a simple way to assay potential gene therapies in mouse models of genetic diseases. In conclusion, this study demonstrates the feasibility of direct DNA injection into the mouse heart and further characterizes the behavior of injected genes. Clearly, the mouse heart efficiently takes up and expresses injected genes in a dose-dependent pattern. Expression of injected naked DNA in the mouse heart is restricted in the localized area of injection and is relatively stable over time. Gene injection into the mouse heart provides a highly efficient and reproducible tool for molecular genetic studies in this well-defined species.
1503
Acknowledgement The authors thank Drs Kass-Eisler, Buttrick and Geenen for their help with the pilot study, and Drs Kass-Eisler, Roopnarine and Buttrick for their critical reading of this manuscript. KL is a recipient of a postdoctoral fellowship of the American Heart Association New York City Affiliate, and LAL is a recipient of NIH grant 5R37HL50560-03.
References A G, J S, J A, D D, W P, W W, W JA, 1991a. Direct gene transfer and expression into rat heart in vivo. New Biol 3: 71–81. A G, D G, L DR, J A, W FS, G A, W JA, D KE, 1991b. Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs. Nature 352: 815–818. B JM, E GI, 1985. Isolation of monoclonal antibodies specific for human c-myc protooncogene product. Mol Cell Biol 5: 3610–3616. B AR, T JE, H JF, 1989. Optimized use of the luciferase assay as a reporter gene in mammalian cell lines. Biotechniques 7: 1116–1122. B PM, K A, K RN, K ML, L LA, 1992. Behavior of genes directly injected into the rat heart in vivo. Circ Res 70: 193–198. D I, F JD, J S, H K, L JS, W JA, 1994. Pharmacological enhancement of in vivo foreign gene expression in muscle. Gene Ther 1: 114–121. D HL, J BJ, 1993. Direct gene transfer into mouse diaphragm. FEBS Let 333: 146–150. D HL, D B, Q B, C J, W R, 1993. Plasmid DNA is superior to viral vectors for direct gene transfer into adult mouse skeletal muscle. Hum Gene Ther 4: 733–740. G D, W L, L G, P JG, H J, I JM, 1993. Direct myocardial transfection in two animal models. Evaluation of parameters affecting gene expression and percutaneous gene delivery. Lab Invest 68: 18–25. G CM, M GT, W MC, P I, H BH, 1982. The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA mediated transfection. Proc Natl Acad Sci USA 79: 6777–6781. K G, A G, 1994. The principles of gene therapy in Duchenne muscular dystrophy. Clin Invest Med 17: 499–509. K R, B P, MN E, K M, L L, 1991. Hormonal modulation of a gene injected into rat heart in vivo. Proc Natl Acad Sci USA 88: 4138–4124. L LA, L JM, 1991. Gene transfer into cardiac myocytes in vivo. Trends Cardiovasc Med 1: 271– 276. L H, P MS, M G, B S, L JM, 1990. Expression of recombinant genes in myocardium in vivo after direct injection of DNA. Circulation 82: 2217–2221.
1504
K. Li et al.
M E, 1993. Regulation of myosin heavy chain genes in the heart. Circulation 87: 1451–1460. N-G B, M V, 1993. Molecular mechanisms of cardiac gene expression. Basic Res Cardiol 88 (Suppl 1): 65–79. R E, C DA, P SE, P TB, A AM, A G, G SH, S M, L D, Y MA, B SM, R GH, 1994. Intradermal gene immunization: the possible role of DNA uptake in the induction of cellular immunity to viruses. Proc Natl Acad Sci USA 91: 9519– 9523. H R, S RJ, S YT, V SF,
M V, N-G B, 1993. Gene injection into canine myocardium as a useful model for studying gene expression in the heart of large mammals. Circ Res 72: 688–695. W JA, M RW, W P, C W, A G, J A, F PL, 1990. Direct gene transfer into mouse muscle in vivo. Science 247: 1465–1468. W JA, W P, A G, J S, J A, C W, 1991. Conditions affecting direct gene transfer into rodent muscle in vivo. Biotechniques 11: 474–485. W JA, C W, A G, W P, 1992. Expression of genes injected into mouse muscle in vivo. Human Mol Genet 1: 53–63.
Direct Gene Transfer into the Mouse Heart Kai Li, Robert E. Welikson, Karen L. Vikstrom and Leslie A. Leinwand Department of Microbiology and Immunology, Albert Einstein College of Medicine, NY, and the Department of Molecular Cellular Developmental Biology, University of Colorado, CO, USA (Received 30 May 1996, accepted in revised form 21 January 1997) K. L, R. E. W, K. L. V L. A. L. Direct Gene Transfer into the Mouse Heart. Journal of Molecular and Cellular Cardiology (1997) 29, 1499–1504. Direct injection of plasmid DNA into the myocardium of several species has been shown to be useful for studying cardiac gene expression. However, despite a better understanding of mouse genetics and the availability of several disease models in mice, gene injection with plasmid DNA into the mouse heart has not been reported. In this study, we demonstrate a simple and reproducible method for gene transfer into the mouse heart via direct injection of plasmid DNA. A firefly luciferase gene, driven by the RSV promoter, was used to quantitatively determine the spatial and temporal characteristics of gene transfer. Luciferase gene expression was stable for 8 weeks and showed a dose-dependent response over a range of 0.3–3 lg of input DNA. Inter-animal variability was low and gene expression was restricted to the left ventricle, near the site of injection. This method was also demonstrated to be suitable for detecting the expression of structural genes under the control of cellular promoters. Immunohistochemistry was used to detect the expression of an epitope-tagged myosin heavy chain driven by a rat a-myosin heavy chain promoter. Thus, naked DNA injection into the mouse heart results in a highly reproducible expression of constructs with either viral or cellular promoters. It is a relatively inexpensive and efficient means of studying cardiac gene regulation in vivo and a useful tool for screening the potential transgenes before generating transgenic mice. 1997 Academic Press Limited
K W: Gene expression; Mouse heart; Gene injection.
Introduction Since the initial demonstration of direct gene injection into mouse skeletal muscle (Wolff et al., 1990), gene injection into various skeletal muscle sites has been studied, and various parameters for optimising gene transfer have been tested. A mouse’s hind limbs, diaphragm, and tongue can each take up and express injected genes (Acsadi et al., 1991a; Wolff et al., 1991; 1992; Davis et al., 1993). Gene transfer into a normal adult skeletal muscle of mouse and rat is quite inefficient, although agents which promote muscle degeneration/regeneration dramatically increase the expression of injected genes (Davis and Jasmin, 1993; Danko et al., 1994). In contrast to skeletal muscle, gene injection into the rat heart is quite
efficient, and results in up to 50-fold higher levels of gene expression than similar experiments in skeletal muscles (Lin et al., 1990; Kitsis et al., 1991; Buttrick et al., 1992). Transfer of naked plasmid DNA into the heart has subsequently been demonstrated in rabbits and dogs (Gal et al., 1993; von Harsdorf et al., 1993). Although the mechanism underlying the uptake of injected genes remains unknown, it appears that this ability is restricted to striated muscles. In rat and rabbit hearts, gene expression after a single DNA injection is generally stable over time. The potential applications of direct gene transfer include potential somatic gene therapy, the analysis of gene regulation in vivo (Kitsis et al., 1991; Buttrick et al., 1992), vaccination (Raz et al., 1994), and hormone replacement therapy (Leinwand and Leiden, 1991).
Please address all correspondence to: Leslie A. Leinwand, Department of Molecular Cellular Developmental Biology, Campus Box 347, University of Colorado, Boulder, CO 80309-0347, USA.
0022–2828/97/051499+06 $25.00/0
mc970389
1997 Academic Press Limited
1500
K. Li et al.
Despite the prevalent use of the mouse in genetic and developmental studies, there are no published reports of DNA injections into the mouse heart. We have developed surgical protocols for mouse cardiac injection and characterized the resulting gene expression. Our data show that gene injection into the mouse heart is highly reproducible, and results in stable gene expression over 8 weeks. When reporter genes were used, expression was dosedependent and restricted to a limited area of the left ventricle. These results are consistent with previous reports of gene injection in rat (Buttrick et al., 1992) and dog hearts (von Harsdorf et al., 1993). The detection of a tagged mammalian contractile protein gene under the control of a cellular promoter, suggests that it may also be possible to evaluate candidate transgenes with this method. The consistency of the results obtained with gene injection into the mouse heart has opened a new avenue for studying mouse cardiac gene expression in vivo, using the most prevalent species in mammalian genetic analysis.
Materials and Methods Adult female CD1 mice were anesthetized with chloral hydrate (0.4 g/kg, i.p.). The animals were placed in a supine position and the upper limbs were taped to a table. Chest skin was cleaned with 70% ethanol and a 1–1.5 cm incision was made along the left side of the sternum. The muscle layers of the chest wall were bluntly dissected to avoid bleeding. The thorax was opened by cutting the rib at the point of the most pronounced cardiac pulsation. While forceps were used to widen the chest, the abdomen and the right side of the chest were pressed to push the heart out of the thoracic cavity. Unless specified, 5 lg of DNA in 15 ll normal saline was injected through a 30-gauge needle into the left ventricular free wall. Following the injection, the heart was placed back into the thoracic cavity and the chest held closed with forceps. After the muscle layers were closed with one suture, the forceps were released. The chest cavity was gently squeezed to expel air out before tightly tying the suture and stapling the skin. Spontaneous respiration was maintained and no mechanical ventilation was needed. Two plasmids were used for this study. The reporter gene, pRSVluc (Buttrick et al., 1992) contains a firefly luciferase cDNA fused to the long terminal repeat of the Rous Sarcoma Virus (RSV LTR) (Gorman et al., 1982). A candidate transgene paa-myc, encoding a myc epitope-tagged rat a-myosin heavy
chain (aMyh) driven by a 2.9 kb rat aMyh promoter (Vikstrom and Leinwand, unpubl) was also used. For luciferase assays, mice were killed by cervical dislocation 1, 2, 4, and 8 weeks after gene injection. The hearts were removed and washed with icecold homogenization buffer (25 m Gly-gly, 15 m MgSO4, 4 m EGTA, and 4 m DTT). The tissue was minced in 0.4 ml homogenization buffer, homogenized for 20 s with a Tissuemizer (Tekmar Company, Cincinnati, OH, USA), and then centrifuged at 5000×g for 20 min (4°C). The supernatant was removed and kept for luciferase assays. Twenty-five percent of the supernatant (100 ll) was mixed with 360 ll reaction buffer (25 m Gly-gly, 15 m MgSO4, 4 m EGTA, 4 m DTT, 15 m KPO4, pH 7.8, and 2 m Na+-ATP). The enzymatic reaction was initiated by injection of 100 ll homogenization buffer containing 0.2 m D-luciferin and light emission was measured for 20 sec (Brasier et al., 1989). The light units measured from the 100 ll of supernatant were expressed as relative luciferase activity. For tissue sectioning and immunostaining, the heart was harvested 5 days after gene injection, flash frozen in liquid nitrogen and stored at −80°C. After embedding in OCT, 5 lm sections were collected every 20 sections and placed on poly--lysine coated slides. The slides were airdried, fixed in acetone (−20°C for 5 min), and then allowed to air dry again. Endogenous peroxidase activity was blocked by incubating in 0.3% hydrogen peroxide in methanol for 30 min at room temperature. All subsequent incubations were done at 37°C in a humidified chamber. After air drying, the slides were blocked by incubating for 30 min with 10% goat serum in phosphate buffered saline (PBS). The sections were then incubated with 9E10.2 culture supernatant (Bishop and Evan, 1985; ATCC CRL 1729) for 30 min. The slides were washed by soaking in three, 5 min changes of PBS and then incubated for 30 min with a 1:200 dilution (in 10% goat serum) of horseradish peroxidase conjugated goat anti-mouse IgG (Bio-Rad, Hercules, CA, USA). Slides were washed as above, and color was developed using the DAB substrate kit (Vector Laboratories, Inc., California, CA, USA), according to the manufacturer’s instructions. Slides were mounted with Airvol (Air Products & Chemical Inc., Allentown, PA, USA). All results are expressed as mean±... A oneway analysis of variance followed by a Student– Newman–Keuls test was used for multiple comparisons. A P value less than 0.05 was used as statistical significance.
1501
Direct Gene Transfer into the Mouse Heart 7
6
*
4
2
0
Relative LUC activity (log)
Relative LUC activity (log)
8
10 3 0.3 1 Dosage of DNA injected ( g)
Figure 1 Relative luciferase (LUC) activity measured in mouse myocardial extracts as a function of DNA dose injected. All values of relative luciferase activity were obtained from 100 ll supernatant. Values are mean±... ∗=P<0.05 as compared to the next group with smaller dose.
6
*
5 4 3 2 1 0
4 6 2 Time after injection (weeks)
8
Figure 2 The level of luciferase (LUC) gene expression as a function of time, where n=6 for each group, except n=7 for the 8 week group. Gene expression was stable the 8 weeks examined. ∗=P<0.05 as compared to the other groups.
Results The survival of mice from the surgery and gene injection was quite high (76%, n=55). After gene injection, death resulting from depressed respiration occurred primarily within the first h. To maximise simplicity, no respirator was used for this surgery. With the luciferase reporter gene, high levels of luciferase activity were detected in all surviving mice using doses of DNA from 0.3 to 10 lg. With the exception of one heart, which probably represents a poor gene injection, the inter-animal variability in luciferase activity was less than one order of magnitude within any group. To determine the optimal DNA dose for gene injection into the mouse heart for the RSV-luciferase construct, four doses of pRSVLuc were tested: 0.3, 1, 3, and 10 lg, all in a volume of 15 ll. Each dose of DNA was injected into five mice, and luciferase activity was assayed 1 week later. As shown in Figure 1, luciferase activity increased in a dose-responsive manner with injections of up to 3 lg DNA, then plateaued. This suggests that to achieve gene expression within the linear range of a RSV LTR driven construct, 3 lg of DNA is the optimal dose. By measuring luciferase levels at various times post-injection, we addressed whether the expression of a reporter gene was stable over time (Fig. 2). The highest level of reporter gene expression was detected 1 week after gene injection (P<0.05 as compared to the other three groups). The expression level decreased slightly thereafter, but remained stable until 8 weeks post injection. The spatial distribution of gene expression in
Relative LUC activity (log)
7 6 5 4 3 2 1 0
LV A + RV Septum Space distribution of reporter gene
Figure 3 Spatial distribution of luciferase (LUC) expression after a single injection into the mouse heart. Three hearts were included in this group and all showed high levels of expression in the left ventricle (LV) and no detectable luciferase activity in atrium (A), right ventricle (RV), and septum.
injected mouse hearts was assessed using the luciferase reporter gene. Luciferase assays were performed on dissected regions of the heart (n=3). As shown in Figure 3, only the left ventricle expressed the reporter gene. Since reporter gene assays, such as luciferase activity, are extremely sensitive, we wished to assess whether expression of genes driven by cellular promoters can be detected following gene injection into the mouse heart. Figure 4 shows the distribution of myc-tagged aMyh protein 1 week after injection of an aMyh-promoter-driven construct into the mouse heart. Immunostaining with a myc-specific monoclonal antibody only revealed gene expression around the needle track in the left ventricle (Fig. 4A). Many cardiac myocytes were positive for the myc-tagged Myh protein in this region. However, there were negative cells within
1502
K. Li et al.
Figure 4 Immunostaining for myc tagged rat a myosin heavy chain expression in the mouse heart 1 week after a 5 lg DNA injection of paamyc plasmid. Dark brown staining indicates myc tagged rat a myosin. An injected heart is shown in (A) and an uninjected is shown in (B). Bar equals 20 lm.
the needle track as well. The highest density of positive cells was seen in the central part of the needle track with fewer positive cells around the periphery of the track. There were no positive cells with the myc antibody in an uninjected mouse heart (Fig. 4B).
Discussion Three observations result from this study. Firstly, we have demonstrated the feasibility of direct gene transfer into the mouse heart. Secondly, we have defined the behavior of genes injected into the mouse heart with respect to the spatial and temporal distribution of gene expression, and thus, provided a range of doses at which a high level expression of injected genes can be obtained. The third contribution of this study, is in demonstrating the ability to evaluate potential transgenes in vivo before producing a transgenic line. This finding is notable, given the inefficiency of transfection into cultured cardiac myocytes. Gene injection into the mouse
heart, thus, provides an easy and useful way for direct gene transfer into a widely used animal species. Gene injection into the mouse heart is fast and simple. Neither extensive sterile technique nor artificial respiration is required. A 77% survival rate and a 100% incidence of positive expression demonstrate its reliability and reproducibility. Any variability of gene expression in the hearts of small mammals, such as the rat and the mouse, is probably due to the quality of the injection. The small size and thin free wall of the mouse heart may significantly contribute to the variability in this animal. Our previous gene injection studies in the rat heart included a vital dye in the DNA solution to guide the injection (Buttrick et al., 1992). In this study, no dye was used since a limited injection volume was allowed. However, we observed evident subepicardial edema immediately following the DNA injection which caused the tissue to change color from red to white. If bleeding was avoided and the chest cavity was not open for longer than 2 min, the survival rate was quite high. From several standpoints, the behavior of genes injected into the mouse heart is similar to that observed in the hearts of rats and dogs (Lin et al., 1990; Acsadi et al., 1991a; Buttrick et al., 1992; von Harsdorf et al., 1993). Our luciferase data indicate that only the left ventricle expressed the injected reporter gene, and the results from paamyc injection further showed that expression of the injected gene was largely restricted to the injection site vicinity. Expression from the RSV-LTR driven construct behaved in a dose-dependent manner in the range of 0.3–3 lg DNA. However, given the quantitatively different promoter activities existing in mammalian cells, it is important to define the optimal dosage for each expression construct injected. Gene expression after plasmid DNA injection is generally stable over time, which is one of the advantages of this method when compared to the more transient gene expression following recombinant adenovirus injection (for review see Leinwand and Leiden, 1991; Nadal-Ginard and Mahdavi, 1993). Finding peak gene expression level 1 week post-injection in the mouse heart is consistent with the findings in rats and dogs (Lin et al., 1990; Kitsis et al., 1991; Buttrick et al., 1992; von Harsdorf et al., 1993). The duration of gene expression in the mouse heart is stable over 8 weeks. The result is similar to findings from previous studies in the rat, showing gene expression for up to 2 months following a single injection (Lin et al., 1990; Kitsis et al., 1991; Buttrick et al., 1992). In
Direct Gene Transfer into the Mouse Heart
addition, very stable gene expression, even over 1 year, in mouse skeletal muscles has been demonstrated after a single plasmid DNA injection (Wolff et al., 1992). Whether gene expression in the mouse heart after DNA injection can persist for the same time period is not addressed in this study, but the relatively stable gene expression we observed over 8 weeks suggests that it may be possible. Compared to other species, direct DNA injection into the mouse heart has several obvious advantages for molecular genetic studies. First, the genetics of the mouse are well understood, and genetic manipulations in the mouse are now performed relatively routinely. A major contribution of DNA injection into animal hearts is in the in vivo analysis of gene regulation. An increasing number of cis- and trans-activating factors have been identified for mammalian genes, generally using transfection of cultured cells (Morkin, 1993). Such approaches for studying cardiac gene regulation have been limited by a lack of permanent cardiac cell lines and an inability to transfect adult primary cardiac myocytes in culture. DNA injection into the mouse heart should become very useful for the study of cardiac gene regulation in vivo, as it has been in the rat (Kitsis et al., 1991; Buttrick et al., 1992). Second, many mouse models for human diseases can be substrates for testing gene therapeutic interventions. One example is the mdx mouse, which is deficient in dystrophin, and has been used to test several approaches in the treatment of muscular dystrophies. These approaches include gene transfer by either direct DNA injection (Acsadi et al., 1991b) or viral vector infection (Karpati and Acsadi, 1994). In addition, following direct DNA injection of a tRNA suppressor gene into the mdx mouse heart, we recently observed a phenotypic correction of cardiac myocytes (Li and Leinwand, in preparation). Since the myocardium takes up DNA more efficiently than other tissue types, gene injection into the mouse heart provides a simple way to assay potential gene therapies in mouse models of genetic diseases. In conclusion, this study demonstrates the feasibility of direct DNA injection into the mouse heart and further characterizes the behavior of injected genes. Clearly, the mouse heart efficiently takes up and expresses injected genes in a dose-dependent pattern. Expression of injected naked DNA in the mouse heart is restricted in the localized area of injection and is relatively stable over time. Gene injection into the mouse heart provides a highly efficient and reproducible tool for molecular genetic studies in this well-defined species.
1503
Acknowledgement The authors thank Drs Kass-Eisler, Buttrick and Geenen for their help with the pilot study, and Drs Kass-Eisler, Roopnarine and Buttrick for their critical reading of this manuscript. KL is a recipient of a postdoctoral fellowship of the American Heart Association New York City Affiliate, and LAL is a recipient of NIH grant 5R37HL50560-03.
References A G, J S, J A, D D, W P, W W, W JA, 1991a. Direct gene transfer and expression into rat heart in vivo. New Biol 3: 71–81. A G, D G, L DR, J A, W FS, G A, W JA, D KE, 1991b. Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs. Nature 352: 815–818. B JM, E GI, 1985. Isolation of monoclonal antibodies specific for human c-myc protooncogene product. Mol Cell Biol 5: 3610–3616. B AR, T JE, H JF, 1989. Optimized use of the luciferase assay as a reporter gene in mammalian cell lines. Biotechniques 7: 1116–1122. B PM, K A, K RN, K ML, L LA, 1992. Behavior of genes directly injected into the rat heart in vivo. Circ Res 70: 193–198. D I, F JD, J S, H K, L JS, W JA, 1994. Pharmacological enhancement of in vivo foreign gene expression in muscle. Gene Ther 1: 114–121. D HL, J BJ, 1993. Direct gene transfer into mouse diaphragm. FEBS Let 333: 146–150. D HL, D B, Q B, C J, W R, 1993. Plasmid DNA is superior to viral vectors for direct gene transfer into adult mouse skeletal muscle. Hum Gene Ther 4: 733–740. G D, W L, L G, P JG, H J, I JM, 1993. Direct myocardial transfection in two animal models. Evaluation of parameters affecting gene expression and percutaneous gene delivery. Lab Invest 68: 18–25. G CM, M GT, W MC, P I, H BH, 1982. The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA mediated transfection. Proc Natl Acad Sci USA 79: 6777–6781. K G, A G, 1994. The principles of gene therapy in Duchenne muscular dystrophy. Clin Invest Med 17: 499–509. K R, B P, MN E, K M, L L, 1991. Hormonal modulation of a gene injected into rat heart in vivo. Proc Natl Acad Sci USA 88: 4138–4124. L LA, L JM, 1991. Gene transfer into cardiac myocytes in vivo. Trends Cardiovasc Med 1: 271– 276. L H, P MS, M G, B S, L JM, 1990. Expression of recombinant genes in myocardium in vivo after direct injection of DNA. Circulation 82: 2217–2221.
1504
K. Li et al.
M E, 1993. Regulation of myosin heavy chain genes in the heart. Circulation 87: 1451–1460. N-G B, M V, 1993. Molecular mechanisms of cardiac gene expression. Basic Res Cardiol 88 (Suppl 1): 65–79. R E, C DA, P SE, P TB, A AM, A G, G SH, S M, L D, Y MA, B SM, R GH, 1994. Intradermal gene immunization: the possible role of DNA uptake in the induction of cellular immunity to viruses. Proc Natl Acad Sci USA 91: 9519– 9523. H R, S RJ, S YT, V SF,
M V, N-G B, 1993. Gene injection into canine myocardium as a useful model for studying gene expression in the heart of large mammals. Circ Res 72: 688–695. W JA, M RW, W P, C W, A G, J A, F PL, 1990. Direct gene transfer into mouse muscle in vivo. Science 247: 1465–1468. W JA, W P, A G, J S, J A, C W, 1991. Conditions affecting direct gene transfer into rodent muscle in vivo. Biotechniques 11: 474–485. W JA, C W, A G, W P, 1992. Expression of genes injected into mouse muscle in vivo. Human Mol Genet 1: 53–63.