Expression of a Human Coxsackie/Adenovirus Receptor Transgene Permits Adenovirus Infection of Primary Lymphocytes 1 Madelyn R. Schmidt, 2 * Brian Piekos, ² Mark S. Cabatingan,* and Robert T. Woodland* Replication-defective adenoviruses are effective vehicles for gene transfer, both for the repair of defective genes and for studies of gene function in primary cells. Many cell types, including lymphocytes, are refractory to adenovirus infection because they lack the Coxsackie/adenovirus receptor (CAR) needed for virus attachment. To extend the advantages of adenovirus-mediated gene transfer to primary lymphoid populations and other cell types lacking endogenous CAR, we produced a mouse that expresses human (h) CAR as a transgene under control of a murine MHC class I promoter. hCAR protein is expressed on T and B lymphocytes from a variety of organs (spleen, lymph node, bone marrow, thymus, and peritoneum). These lymphocytes are susceptible to adenovirus infection, as demonstrated by reporter green fluorescent protein gene expression, with the fraction of expressing cells as high as 70%. Some lymphocyte subpopulations required stimulation subsequent to adenovirus infection for reporter expression. This activation requirement is a restriction imposed by the promoter used in the adenovirus construct. In sub- populations requiring activation, the elongation factor 1 promoter was far superior to a hCMV promoter for directing green fluorescent protein production. We also find that hCAR mRNA is produced in nonlymphoid tissues from all founder lines, including tissues that do not express endogenous murine CAR, suggesting the opportunity for effecting gene delivery to and testing gene function in a wide variety of primary cell types previously resistant to gene transfer. The Journal of Immunology, 2000, 165: 4112– 4119. T he transfer of genes into mammalian cells using viral vec- tors has provided a powerful tool for correcting genetic defects and for studying the involvement of specific genes in cellular processes such as proliferation, differentiation, cell death, and survival. Replication-defective adenovirus vectors have many advantages as vehicles for gene transfer (1– 4). Stocks of modified adenovirus are readily produced in high titer, superinfec- tion with adenoviruses encoding different genes is possible, and gene expression is robust and of relatively long duration. Unlike most retrovirus vectors, which require cycling target cells for vec- tor integration and efficient gene expression, adenovirus infection and subsequent gene expression can occur in quiescent cells (5, 6). Adenovirus is primarily maintained in the nuclei of infected cells, in association with the nuclear matrix, without integration (7). This precludes insertional mutagenesis of the host genome and renders virally encoded genes refractory to silencing as a consequence of insertion adjacent to negative regulatory elements. Extrachromo- somal replication of the adenovirus (AdV) 3 genome can be achieved in modified vectors, without the production of infectious particles, potentially allowing durable gene expression in replicat- ing cells (8). Efficient adenovirus infection requires the presence of the Cox- sackie/adenovirus receptor (CAR) for virus attachment to the cell surface and an integrin coreceptor,  v  3 or  v  5 , which promotes virus internalization (9 –12). Both human (h) and murine (m) genes for CAR have been identified and, although they have a different tissue expression pattern, either will allow infection by human ad- enovirus when transfected into CAR-negative cell lines (9, 10, 13, 14). Unfortunately, in the mouse, many interesting target tissues, including cells of hemopoietic origin, do not express CAR, limit- ing the use of recombinant adenoviruses for studies of gene func- tion or replacement in well-characterized murine model systems of disease, particularly immunodeficiencies (9, 10, 13, 15). To circumvent this problem, we generated transgenic mice that express hCAR. Transgene expression is controlled by the murine MHC class I H-2K b promoter/enhancer, which has been shown to support nearly ubiquitous and constitutive transgene expression, including cells of hemopoietic origin (16 –18). hCAR protein was readily detected on the surface of B and T lymphocytes from spleen, lymph nodes, bone marrow, thymus, and peritoneum. hCAR expression was sufficient to permit adenovirus infection and subsequent reporter gene expression in primary lymphocyte pop- ulations that were refractory to adenovirus infection when isolated from wild-type mice. We also found hCAR mRNA in tissues that do not express endogenous mCAR, demonstrating the potential to use adenovirus as a vehicle for gene delivery to primary cell types not previously susceptible to this method of gene transfer. Materials and Methods Cloning and transgene detection A cDNA clone encoding hCAR/pcDNA1 was generously provided by Dr. Robert Finberg, Dana-Farber Cancer Institute (Boston, MA) (9, 10). The hCAR plasmid was digested with EcoRI releasing a 2.5-kb fragment con- taining the full hCAR coding region. This fragment was blunted, NotI *Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01655; and ² Biogen, Cambridge, MA 02142 Received for publication May 3, 2000. Accepted for publication July 12, 2000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work is sponsored by a grant to M.R.S. from the Worcester Foundation for Biomedical Research Annual Research Fund Innovation Grant, and to R.T.W. from the National Institutes of Health, AI41054 and AI30890, and an Institutional Diabetes and Endocrinology Research Center Grant from the National Institutes of Health, DK32520. 2 Address correspondence and reprint requests to Dr. Madelyn R. Schmidt, Department of Molecular Genetics and Microbiology, University of Massachu- setts Medical School, 55 Lake Avenue North, Worcester, MA 01655. E-mail address: Madelyn.Schmidt@umassmed.edu 3 Abbreviations used in this paper: AdV, adenovirus V; -gal, -galactosidase; BSS, balanced salt solution; CAR, Coxsackie/adenovirus receptor; EF1, elongation factor 1; GFP, green fluorescent protein; h, human; LTR, long terminal repeat; m, murine; MFV, mean fluorescent value; moi, multiplicity of infection; PBTL, peripheral blood T lymphocyte. 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