Protection Against Hyperacute Xenograft Rejection of Transgenic Rat Hearts Expressing Human Decay Accelerating Factor (DAF) Transplanted into Primates

Abstract

Background: Production of transgenic pigs for multiple transgenes is part of a potential strategy to prevent immunological events involved in xenograft rejection. Use of a genetically engineerable rodent as a donor in primates could allow testing in vivo of the effects of different transgenes on controling xenograft rejection. As a first step in the development of a donor containing multiple transgenes, transgenic rats for human decay-accelerating factor (DAF) were used as heart donors to test their resistence against complement (C)-mediated rejection by non-human primates. Materials and Methods: Transgenic rats were generated by using a construct containing the human DAF cDNA under the transcriptional control of the endothelial cell (EC)-specific human ICAM-2 promoter. DAF expression was evaluated by immnunohistology and by FACS analysis of purified ECs. Resistance of transgenic hearts against C-mediated damage was evaluated by ex vivo perfusion with human serum and by transplantation into cynomolgus monkeys. Results: Immunohistological analysis of DAF expression in several organs from two transgenic lines showed uniform expression on the endothelium of all blood vessels. ECs purified from transgenic hearts showed 50% DAF expression compared to human ECs and >70% reduction of C-dependent cell lysis compared to control rat ECs. Hemizygous transgenic hearts perfused with human serum showed normal function for >60 min vs. 11.2 ± 1.7 min in controls. Hemior homozygous transgenic hearts transplanted into cynomolgus monkeys showed longer survival (15.2 ± 7 min and >4.5 hr, respectively) than controls (5.5 ± 1.4 min). In contrast to hyperacutely rejected control hearts, rejected homozygous DAF hearts showed signs of acute vascular rejection (AVR) characterized by edema, hemorrhage, and an intense PMN infiltration. Conclusions: We demonstrate that endothelialspecific DAF expression increased heart transplant survival in a rat-to-primate model of xenotransplantation. This will aid in the analysis of AVR and of new genes that may inhibit this form of rejection, thus helping to define strategies for the production of transgenic pigs.

In vivo cell lineage analysis in cyproterone acetate-treated rat liver using genetic labeling of hepatocytes

“…LacZ transgenic rats were generated by microinjection of a DNA construct encoding the cytoplasmic form of E. coli ␤-galactosidase driven by the human immunodeficiency virus-long terminal repeat (HIV-LTR) promoter 20 into eggs from the SpragueDawley strain according to previously described protocols. 21,22 All animals received food and drinking water ad libitum throughout the experimental period.…”

lacZTransgenic Rats Tolerant forβ-Galactosidase: Recipients for Gene Transfer Studies UsinglacZas a Reporter Gene

“…Transgenic rats were produced following previously published procedures (Charreau et al, 1996(Charreau et al, , 1999. Embryos were microinjected with a DNA construct (2 mg/ml), previously used to generate transgenic mice (Cavard et al, 1990) (kindly provided by P. Briand, Institut Cochin, Paris, France).…”

“…Because of its bigger size, the rat is more accessible than the mouse to microsurgery, multiple tissue and organ sampling, injection of substances into the brain followed by precise neuroanatomic localization (in mice there is lack of resolution through diffusion seen in a smaller brain), and analysis of organ function ex vivo (e.g., heart perfusion). Furthermore, a large body of physiologic data and disease models (Gill et al, 1989) as well as transgenic technology (Charreau et al, 1996(Charreau et al, , 1999 are available in the rat. Rats transgenic for reporter genes and with immune tolerance toward the transgene could represent a useful tool for the gene transfer field.…”

Production of Transgenic Rats

“…Human apo A-I transgenics were used in the study of lipid metabolism and treatment of cholestasis (Chisholm et al, 2000). Polyamine metabolism has been analyzed in spermidine/spermine N(1)-acetyltransferase transgenics (Alhonen et al, 2000), and cardiac transplant models were developed where transgenic rat hearts were transplanted into primates, allowing the assessment of hyperacute rejection (Charreau et al, 1999).…”