The human cannabinoid G protein-coupled receptors (GPCRs) CB1 and CB2 mediate the functional responses to the endocannabinoids anandamide and 2-arachidonyl glycerol (2-AG), as well as the widely consumed plant (phyto)cannabinoid Δ9-tetrahydrocannabinol (THC)1. The cannabinoid receptors have been the targets of intensive drug discovery efforts due to the therapeutic potential of modulators for controlling pain2, epilepsy3, obesity4, and other maladies. While much progress has recently been made in understanding the biophysical properties of GPCRs, investigations of the molecular mechanisms of the cannabinoids and their receptors have lacked high-resolution structural data. We used GPCR engineering and lipidic cubic phase (LCP) crystallization to determine the structure of the human CB1 receptor bound to the inhibitor taranabant at 2.6 Å resolution. CB1's extracellular surface, including the highly conserved membrane-proximal amino-terminal (N-terminal) region, is distinct from other lipid-activated GPCRs and forms a critical part of the ligand binding pocket. Docking studies further demonstrate how this same pocket may accommodate the cannabinoid agonist THC. Our CB1 structure provides an atomic framework for studying cannabinoid receptor function, and will aid the design and optimization of cannabinoid system modulators for therapeutic ends.

The use of a transgenic line of rats that express enhanced GFP (EGFP) exclusively in the germ line has allowed a separation of feeder layers and contaminating testis somatic cells from germ cells and the identification of a set of spermatogonial stem cell marker transcripts. With these molecular markers as a guide, we have now devised culture conditions where rat spermatogonial stem cells renew and proliferate in culture with a doubling time between 3 and 4 days. The marker transcripts increase in relative abundance as a function of time in culture, and the stem cells retain competency to colonize and develop into spermatids after transplantation to the testes of recipient rats. The cells also remain euploid after at least 12 passages. Cell lines could be isolated and cryopreserved and, upon subsequent thawing, continue to self renew. Transfection of the spermatogonial stem cells with a plasmid containing the neomycin phosphotransferase (neo) selectable marker resulted in selection of G418-resistant cell lines that effectively colonize recipient testes, suggesting that gene targeting is now feasible in the rat.germ line ͉ spermatogenesis ͉ gene targeting ͉ enhanced GFP fluorescence T he laboratory rat represents one of the most comprehensively studied mammalian species, with described use in Ͼ1 million publications in a wide range of medically relevant areas. Qualities such as size, fecundity, behavior, ease of surgical techniques, tissue sampling, and general laboratory management have contributed to its popularity (1-3). However, a failure to develop technology to produce rat genetic models through gene targeting has resulted in the mouse becoming a widely popular animal model.Although mouse embryonic stem (ES) cells renew with a sense of immortality, primitive hemapoietic stem cells self renew ineffectively and for only a short period in vitro (4, 5). ES cells from species other than the mouse or human fail to generally self renew effectively and also lose pluripotency in culture (6). We and others have not succeeded in culturing pluripotent ES cells from the rat; however, if spermatogonial stem cells could be cultured under conditions where they self renewed and expanded in numbers, conceivably they could also be genetically modified in vitro in much the same manner as seen with mouse ES cells. This alternative to the use of genetically modified ES cells would result in direct germ line transfer and an escape from the intervening formation of a mosaic animal. After appropriate selection of gene-targeted cells in culture, the chosen spermatogonial stem cells could either be induced to differentiate to the haploid stage in vitro or transplanted to the testes of recipient rats to allow development to the haploid stage. In either case, intracytoplasmic sperm injection into the egg would result in transmission of the genetically modified information (7,8).We now show that genetically marked spermatogonial stem cells from transgenic germ-cell-specific EGFP (GCS-EGFP) rats (9) can be placed under defined culture co...

Primary cultures of rat spermatogenic cells that did not bind to collagen matrices were able to colonize and form mature spermatozoa when transferred to testes of recipient males. Up to 73% of the progeny from matings with recipient males were derived from the transferred spermatogenic cells. Subsequently, two populations of germ cells were obtained by selection on laminin matrices. Both populations expressed the spermatogenic cell marker, DAZL, but not the somatic cell marker, vimentin. The cells that bound to laminin represented ≈5% of the total population and were greatly enriched in ability to colonize a recipient testis, suggesting an enrichment in germ-line stem cells. The colonization potential was maintained for at least 7 days in culture. These cells were subsequently transduced with a lentiviral enhanced GFP reporter vector and then transferred to WT recipient males. After mating, 26 of 44 pups were derived from the cultured donor germ cells, and 13 pups carried the lentiviral transgene. Based on Southern analysis, the transgene was integrated at a different genetic locus in each animal and was transmitted to ≈50% of pups in the F 2 generation. Thus, by using these procedures, ≈30% of pups in the F 1 generation inherited and stably transmitted a lentiviral transgene that integrated at various genomic sites.