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In Vivo Promoter Activity and Transgene Expression in Mammalian Somatic Tissues Evaluated by Using Particle Bombardment LIANG CHENG, PAMELA R

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In Vivo Promoter Activity and Transgene Expression in Mammalian Somatic Tissues Evaluated by Using Particle Bombardment LIANG CHENG, PAMELA R Proc. Natl. Acad. Sci. USA Vol. 90, pp. 4455-4459, May 1993 Genetics In vivo promoter activity and transgene expression in mammalian somatic tissues evaluated by using particle bombardment LIANG CHENG, PAMELA R. ZIEGELHOFFER, AND NING-SUN YANG* Department of Mammalian Genetics, Agracetus, Inc., 8520 University Green, Middleton, WI 53562 Communicated by C. C. Tan, February 8, 1993 ABSTRACT The particle bombardment method of gene extracts and at the cellular level from various reporter genes transfer provides an alternative approach for analysis ofin vivo bombarded into the skin and liver tissues ofrats and mice (10, promoter activity and transgene expression. Transient expres- 11). The Accell bombardment method, which utilizes an sion ofthe firefly luciferase gene from five viral and five ceUlular electric discharge device to accelerate DNA-coated micro- promoters was assessed after in vivo gene transfer using this scopic gold particles into target tissues, requires minimal method. The relative strengths of these promoters were quan- manipulation of the target organs of test animals, and results titatively determined in five different rat tissues: skin epider- can be readily obtained after gene transfer. Since particle mis, dermis, muscle, liver, and pancreas. Cytomegalovirus bombardment utilizes a physical force for gene delivery, it is immediate early enhancer/promoter activity was consistently apparently not dependent on cell membrane properties ofthe the highest in each tissue, whereas other promoters displayed various target cell types (10-12). tissue-specific preferences. In liver, the mouse phospho- In this study, we evaluate its use for in vivo promoter enolpyruvate carboxykinase and metaflothionein promoters characterization in various rat somatic tissues, for long-term were stimulated in vivo by inducing agents at 1 and 5 days transgene expression, and for application to other mamma- posttransfection. In dermis, sustained luciferase activity was lian systems. Future applications to gene transfer and gene observed for over 1.5 years after gene delivery. In vivo trans- therapy are discussed. gene expression was also detected in bombarded mouse, rabbit, and rhesus monkey tissues. These results suggest that particle bombardment provides an effective system for studies ofin vivo MATERIALS AND METHODS gene transfer and gene therapy. Plasmids. A series of vectors expressing the firefly lu- ciferase (luc) gene under the regulatory control of various Currently, the expression patterns of most mammalian trans- viral and cellular gene promoters was used. pNASSluc (no gene constructs are analyzed by using cell culture systems (1, promoter) and pADluc (containing the adenovirus 2 major 2), which may not reflect the behavior of specific promoters late promoter) were made by replacing the lacZ gene from or transgenes under in vivo conditions (3). With the recent pNASSf3 and pAD,B (Clontech) with the luciferase gene as a progress in gene therapy research (4, 5), there is a need for Not I fragment from pCMVluc. Plasmids pCMVluc, pRSV- an efficient gene delivery and assay system to characterize luc, pSVluc, and pMLVluc, which contain the cytomegalo- promoter-therapeutic gene constructs in vivo, allowing ex- virus (CMV) immediate early gene enhancer/promoter, Rous amination of transgene expression levels and physiological sarcoma virus (RSV) long terminal repeat, Simian virus 40 effects. It is also highly desirable that this system be appli- (SV40) enhancer/promoter, and the U3 cable to the early region of the many different somatic tissues and mammalian murine leukemia virus (MLV) long terminal repeat, respec- species pertinent to gene therapy experiments. tively, have been described (13). Plasmids pmMTluc, pPEP- One of the few systems currently being used to analyze luc, pBLGluc, and pPLluc, which contain the mouse metal- promoter and transgene activity in vivo is the transgenic lothionein mouse (6, 7). Although this system has proven to be a (mMT) gene promoter (14), mouse phosphoenol- valuable and powerful approach for studying transgene ex- pyruvate carboxykinase (PEP) gene promoter (15), bovine pression in vivo, it is, however, limited in its 13-lactoglobulin (BLG) gene promoter (16), and bovine pro- ability to provide lactin (PL) gene promoter (17), respectively, were con- a convenient and quantitative analysis of transgene activity. structed When compared with the same tissue by inserting the isolated promoters into theXho I site type, extremely vari- of pNASSluc. pCMV,3-gal consists of the CMV promoter able levels of transgene activity were observed among dif- of ferent lines of transgenic mice created using the same pro- driving expression 3-galactosidase ((3gal) (10). The pPGK- moter-gene construct luc plasmid expressing the luciferase gene from the mouse (8). This is because transgene expres- phosphoglycerate kinase (PGK) gene promoter, a gift from sion levels have been shown to be readily influenced by J. A. Wolff position effects at the site of integration on host chromo- (University of Wisconsin, Madison), was previ- somes (9). Techniques for generating transgenic mice are not ously referred to as pKJ-1-Lux (18). yet routinely applicable to other mammalian systems and are In Vivo Bombardment of Various Organs. Particle bom- also costly, laborious, and time-consuming. These various bardment was performed as described using the Accell gene limitations make it impractical as a routine assay system for delivery system (Agracetus, Middleton, WI) (10). Briefly, in vivo transgene expression in targeted somatic tissues of adult male Holtzman rats were anesthetized by i.m. injection test animals. of a mixture of ketamine (70 mg/kg of body weight) and We have previously demonstrated that the particle bom- xylazine (6 mg/kg of body weight). Rabbits and mice were bardment method of gene transfer is effective in various anesthetized by i.m. injection of a mixture of ketamine (50 somatic tissues in vivo and ex vivo (10). High levels of mg/kg of body weight) and xylazine (10 mg/kg of body transgene activity have been readily detected both in tissue weight). Rhesus monkeys were anesthetized with ketamine Abbreviations: CMV, cytomegalovirus; f-gal, /8-galactosidase; The publication costs of this article were defrayed in part by page charge mMT, mouse metallothionein; PEP, phosphoenolpyruvate carboxy- payment. This article must therefore be hereby marked "advertisement" kinase; PGK, phosphoglycerate kinase; RLU, relative light unit. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 4455 Downloaded by guest on September 29, 2021 4456 Genetics: Cheng et al. Proc. Natl. Acad. Sci. USA 90 (1993) (10-15 mg/kg, i.m.). After induction of anesthesia, abdom- The abdominal skin epidermis and dermis, muscle, liver, inal hair was removed by treatment with Nair (Carter- and pancreas tissues of anesthetized animals were exposed Wallace, New York), and the exposed skin was sterilized and bombarded over a uniform target area (3.24 cm2). Tran- with povidone-iodine and rinsed with 70% ethanol. Abdom- sient luciferase activity was found to peak around 16 hr after inal muscle and dermis targets were exposed s.c. by making bombardment for all tested tissues; thus, uniform size tissue a 3-cm-long incision in the abdomen. Abdominal dermis samples from the bombarded sites (3.24 cm2 x 1.5 mm thick; tissue was separated from the underlying abdominal muscle -0.5 g) were obtained from sacrificed test animals at this time tissue with blunt-ended forceps. The thin fascia layer of point. Tissue extracts from these samples were assayed for dermis was carefully removed using fine tweezers. The liver luciferase activity, with the specific activity for luciferase and pancreas were bombarded by making a 2-cm incision into gene expression defined as relative light units (RLUs) per the abdominal cavity and exposing the organ. Each targeted uniform tissue sample. rat tissue site was then bombarded in situ at 21 kV with 0.8 Fig. 1 shows that wide variations in specific luciferase ,ug of plasmid DNA coated onto 0.32 mg of gold particles (1-3 activity can be observed between different promoters in test ,m in diameter). Details of Accell particle bombardment tissues. The CMV immediate early enhancer/promoter con- techniques for DNA loading capacity and effects of discharge sistently displayed high specific activities relative to other voltage have been described (10, 12). After bombardment, tested promoters in all tissues. The specific activities of the the muscle tissue layer was sutured, and the skin was closed other promoters, relative to each other, varied with tissue with stainless steel clips. type. Two mouse cellular promoters, from the PGK gene and In Vivo Induction of Luciferase Transgene Expression. Rat the PEP gene, were as active as the CMV promoter in dermis livers were bombarded in situ with pPEPluc or pmMTluc tissue (Fig. 1A). The mMT promoter was very active in DNA. After 1 or 5 days, pPEPluc-bombarded animals were epidermis (Fig. 1B); it expressed a level similar to that of the given two i.p. injections of N6,02'-dibutyryladenosine 3',5'- CMV promoter. The Rous sarcoma virus long terminal repeat cyclic monophosphate (dibutyryl-cAMP) and theophylline and simian virus 40 early region enhancer/promoter, two (each at 30 mg/kg of body weight) over a 2-hr interval. The commonly used viral promoters, expressed luciferase in pmMTluc-bombarded rats were injected with ZnSO4 (20 muscle, liver, and pancreas at higher levels than any of the mg/kg) following the same injection regime for induction. cellular promoters tested (Fig. 1 C-E). The adenovirus major Control (uninduced) rats were injected with a 0.9% saline late promoter was as active as the other viral promoters in solution. Tissue samples corresponding to the bombarded muscle tissue (Fig. 1C), but was relatively ineffective in other sites were collected 4 hr after the first injection and assayed tested tissues. The commonly used murine leukemia virus for luciferase activity. promoter consistently displayed the lowest activity among Tissue Extraction and Enzyme Assays.
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