Cell Category

In this study, we successfully produced a somatically cloned transgenic piglet using recombinant cells obtained after gene transfer of a transgene (carrying both EGFP and pac expression units) and subsequent in vitro selection with a low concentration (2 ^g/ml) of puromycin. In contrast, no piglets were born when the conventional method, in which a period […]

Puromycin Sensitivity Assay for Fetal Porcine Somatic Cells To determine the optimal concentration of puromycin for the selection of pac-transfected cells, a puromycin resistance test was performed by culturing fetal porcine somatic cells in a medium containing 0.5-6.0 |xg/ml of puromycin for 7 days. No surviving colonies were obtained when exposed to puromycin at concentrations […]

Protocols for the use of animals in this study were approved by the Animal Care Committee of the National Institute of Agrobiological Sciences. Construction of Vectors The transgene, termed pEGFPac (Fig. 1), was constructed by inserting CAG-EGFP fragment into pPGK-pac-p(A) cassettes. Briefly, pCAG-EGFP, an EGFP-expression cassette, was first constructed according to Okabe et al.. The […]

Somatic nuclear transfer has been a useful technique for the production of genetically engineered domestic animals including gene-targeted and transgenic animals. To date, transgenic sheep, pigs, and cattle and gene-targeted sheep and pigs have been successfully produced by nuclear transfer employing transfected somatic cells as nuclear donors. These genetically defined cloned animals may be used […]

The addition of DDE to human granulosa cells caused [Ca2+]cyt elevations, which were detected as increases in the baseline 340:380 nm ratios, and oscillations, which were recorded as increased frequency of peak ratios. In previous studies with porcine granulosa cells, the major effect of DDE was an inhibtion of progesterone production. However, the concentrations of […]

Ca2+ Changes in Response to DDE Figure 1 shows the [Ca2+]cyt changes that occurred when the granulosa cells were exposed to an environmentally relevant dose of DDE (100 ng/ml). This figure is representative of three separate experiments using cells from a single patient in each experiment. Six probes were placed, two on each of three […]

Chemicals and Reagents Human pituitary FSH (EC-232-662-3, lot 092K1105, 7000 IU IRP 68/ 140 per mg), ionomycin, ATP, and DDE were purchased from Sigma-Aldrich Chemicals (Oakville, ON, Canada). Human chorionic gonadotropin (lot B18856, 3050 IU/mg, WHO 1st IRP 75/551) was purchased from Calbiochem (La Jolla, CA). All tissue culture supplies were purchased from Gibco Life […]