in vivo and in vitro gene transfer to mammalian somatic cells by
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Proc. Natl. Acad. Sci. USAVol. 87, pp. 9568-9572, December 1990Genetics
In vivo and in vitro gene transfer to mammalian somatic cells byparticle bombardment
NING-SUN YANG*, JOE BURKHOLDER, BETH ROBERTS, BRIAN MARTINELL, AND DENNIS MCCABE
Department of Mammalian Genetics, Agracetus, 8520 University Green, Middleton, WI 53562
Communicated by Winston J. Brill, September 24, 1990 (receivedfor review August 28, 1990)
ABSTRACT Chimeric chloramphenicol acetyltransferaseand P-galactosidase marker genes were coated onto fine goldparticles and used to bombard a variety of mammalian tissuesand cells. Transient expression of the genes was obtained inliver, skin, and muscle tissues of rat and mouse bombarded invivo. Similar results were obtained with freshly isolated ductalsegments of rat and human mammary glands and primarycultures derived from these explants. Gene transfer and tran-sient expression were also observed in eight human cell culturelines, including cells of epithelial, endothelial, fibroblast, andlymphocyte origin. Using CHO and MCF-7 cell cultures asmodels, we obtained stable gene transfer at frequencies of 1.7x 10-3 and 6 x 10-4, respectively. The particle bombardmenttechnology thus provides a useful means to transfer foreigngenes into a variety of mammalian somatic cell systems. Themethod is applicable to tissues in vivo as well as to isolated cellsin culture and has proven effective with all cell or tissue typestested thus far. This technology may therefore prove to beapplicable in various aspects of gene therapy.
Direct transfer of biologically active foreign genes into mam-malian somatic tissues or organs in vivo is an attractivestrategy for gene therapy and the delivery of therapeuticproteins and peptides (1, 2). A number of methods have beenreported to achieve this goal with varying degrees of success.These include the direct injection of naked plasmid DNA intomuscles (2), delivery of DNA complexed with specific pro-tein carriers (3), coprecipitation of DNA with calcium phos-phate (4), encapsulation of DNA in various forms of lipo-somes (5, 6), and in vivo infection using cloned retroviralvectors (7, 8). We previously developed particle bombard-ment technology to accomplish genetic transformation ofcrop plants (9-14). In this report, we assess the possibility ofdirect in vivo gene transfer into mammalian somatic tissuesby using this developing technology.We have previously described the design of a high-voltage
electric discharge device to be used for the acceleration ofDNA-coated gold particles into living cells (10, 11). With thisdevice a motive force is generated to accelerate the DNA-coated gold particles to high velocity, enabling efficientpenetration of target organs, tissues, or single cells. We showthat our particle bombardment technology can efficientlydeliver foreign genes into liver, skin, and muscle tissues of rator mouse in vivo. Expression of reporter genes in animals canbe readily detected in the target organs at 1-2 days after genetransfer.
This technology can also be applied to solid tissue ex-plants, organ slices, or organoids in vitro. Bombarded tissuematerials can be monitored in vitro for specific experimentalpurposes or transplanted back into the body for gene therapyor other clinical purposes (1, 15). We show that the particle-
bombardment method can effect gene transfer in freshlyisolated mammary gland ductal segments and their derivedprimary cultures, as well as in various human cell lines. In thelatter case, stable gene transfer was demonstrated in twoestablished cell culture lines at a high transfection frequency.Our results suggest that this gene transfer method is appli-cable to a variety of different mammalian somatic cell sys-tems.
MATERIALS AND METHODSParticle Bombardment Device and DNA Delivery. The pro-
cedure for utilizing the particle bombardment device has beendiscussed in detail elsewhere (10) and involves several basicsteps. Plasmid DNA in solution is first precipitated onto goldbeads, which are chemically inert in most, if not all, biologicalsystems. The DNA-coated particles are then resuspended inethanol and evenly deposited onto a thin Mylar film. TheMylar sheet is then placed adjacent to two closely spacedelectrodes, where an electric arc, generated by a high-voltagedischarge, provides the motive force. The force acceleratesthe DNA-coated gold particles to high velocity, enablingefficient penetration of target organs, tissues, or single cells.With our particle bombardment device we can finely tune andadjust the velocity and resulting distribution of gold particlesin various target tissues by varying the discharge voltage(3-25 kV), gold bead density, and bead size. For this study,1- to 3-em gold beads at a density of 2 x 106 beads (0.1 mg)per cm were routinely used, with the velocity of the beadsadjusted by using different discharge voltages as indicated.Plasmid DNA samples were loaded onto beads at 1 ,ug ofDNA per mg of beads, corresponding to 10,000 copies of a5-kilobase (kb) DNA molecule per gold particle (1-3 ,um).
Plasmids. Five plasmid DNA constructions, RSV-CAT,RSV-Neo, RSV-Lux, MMTV-f-Gal, and CMV-f3-Gal, wereused as reporter genes. The RSV-CAT gene constructioncontains the Rous sarcoma virus (RSV) long terminal repeat(LTR) promoter and the chloramphenicol acetyltransferase(CAT) coding sequence (16). RSV-Neo contains the RSVLTR promoter and the neomycin phosphotransferase II(NPT II) coding sequence (17). RSV-Lux contains the RSVLTR promoter and the firefly luciferase coding sequence (2).CMV-P3-Gal contains the cytomegalovirus (CMV) immediateearly promoter and Escherichia coli j-galactosidase (p-Gal)coding sequence (18). The MMTV-f3-Gal contains an RSVLTR enhancer, a mouse mammary tumor virus (MMTV)promoter (19, 20), and the 8-galactosidase coding sequence.The MMTV-,B-Gal expression vector was constructed by T.Mulcahy (University ofWisconsin, Madison) by inserting theE. coli f3-galactosidase gene coding sequence into the pMAM-
Abbreviations: RSV, Rous sarcoma virus; MMTV, mouse mammarytumor virus; CMV, cytomegalovirus; LTR, long terminal repeat;CAT, chloramphenicol acetyltransferase; NPT 11, neomycin phos-photransferase 11.*To whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. 1734 solely to indicate this fact.
Proc. Natl. Acad. Sci. USA 87 (1990) 9569
neo vector obtained from Clontech. The coding sequences forall of these reporter genes have a simian virus 40 polyade-nylylation signal sequence at the 3' terminus.In Vivo Bombardment ofVarious Organs. To perform in situ
bombardment of liver tissues in live animals, we surgicallyopened the rat abdominal cavity and exposed the tip ofa liverlobe as the target site. At energy settings of 10-14 kV bleedingwas not visible to the naked eye. Small hemorrhage spotswere detectable under low-magnification microscopy. Theextent of these spots indicated tissue damage from blastingwas minimal. After the in vivo bombardment the incisionswere closed with sutures. After surgery the animals recov-ered from anesthesia in an hour and behaved normally.Forty-eight hours later the animals were sacrificed. Thebombarded portion of liver lobe was excised, tissue wasextracted, and CAT activity was assayed (21). For in vivobombardment of mouse skin tissue, hair was removed fromthe abdomen by treatment with Nair (Carter-Wallace, NewYork) and the exposed epidermal tissue was bombarded at 10kV. For bombarding mouse muscle tissue, abdominal musclewas surgically separated from the skin tissue layer, theexposed muscle was bombarded at 15 or 18 kV, and theanimals were sutured closed. The mice were sacrificed 24-48hr after blasting, and the bombarded organs were excised.Cell-free extracts were prepared and assayed for CAT activ-ity.In Vitro Bombardment of Tissue Cultures. Ductal segments
ofhuman and rat mammary gland were isolated from excisedmammary tissues by collagenase and hyaluronidase digestionof surrounding stroma tissue (22, 23). Mammary gland tissuesand the derived cultures were generously provided by M.Gould (University of Wisconsin, Madison). MCF-7 cells, ahuman mammary carcinoma cell line, and other human celllines were obtained from the American Type Culture Col-lection and were cultured under conditions recommended bythat supplier (24). In vitro tissue samples, including mammarygland organoids, suspended cells, or monolayer cell cultures,were placed onto a Petri dish substratum and bombarded atappropriate voltage settings as indicated. Two days later,they were harvested from cultures and assayed for markergene expression. When MMTV-,B-Gal was used as a reportergene for bombardment, target tissue or cell cultures weregrown in corresponding culture media containing dexamethe-sone at 2 ,tg/ml, for induction of MMTV promoter activity(19).
Stable Transfection of MCF-7 and CHO Cells. MCF-7 andCHO (Chinese hamster ovary) cells were transfected bybombarding cells at 7 kV, 1 Ag of RSV-Neo DNA per mg ofbeads, 0.1 mg of beads per cm2. Bombarded cells werecultured for 1 day, transferred, and cultured for additional 2days, and then grown under G418 (GIBCO) selection at 200,ug/ml. Antibiotic-resistant colonies appeared between 10and 20 days after selection. A number ofcolonies were scoredmicroscopically to obtain transfection frequency, and trans-fected cells were pooled by trypsinization. Stably transfectedMCF-7 cells were cultured and subcloned for an additional 50passages (-1 year) under G418 selection. CHO cell cloneswere cultured with G418 for 5-10 passages.
Tissue Extraction and Enzyme Assays. Bombarded liver,skin, muscle, and mammary tissues were extracted with CATbuffer (21) and cent