Rikio Niki

Production of transgenic miniature pigs by pronuclear microinjection

Miniature pig is an attractive animal for a wide range of research fields, such as medicine and pharmacology, because of its small size, the possibility of breeding it under minimum environmental controls and the physiology that is potentially similar to that of human. Although transgenic technology is useful for the analysis of gene function and for the development of model animals for various diseases, there have not yet been any reports on producing transgenic miniature pig. This study is the first successful report concerning the production of transgenic miniature pig by pronuclear microinjection. The huntingtin gene cloned from miniature pig, which is a homologue of candidate gene for Huntingtons disease, connected with rat neuron-specific enolase promoter region, was injected into a pronucleus of fertilized eggs with micromanipulator. The eggs were transferred into the oviduct of recipient miniature pigs, whose estrus cycles were previously synchronized with a progesterone analogue. A total of 402 injected eggs from 171 donors were transferred to 23 synchronized recipients. Sixteen of them maintained pregnancy and delivered 65 young, and one resulted in abortion. Five of the 68 offspring (three of which were aborted) were determined to have transgene by PCR and Southern analysis. The overall rate of transgenic production was 1.24% (transgenic/injected eggs). This study provides the first success and useful information regarding production of transgenic miniature pig for biomedical research.

N-(2-Carboxyphenyl)-4-chloroanthranilic acid disodium salt: prevention of autoimmune kidney disease in NZB/NZW F1 hybrid mice

NZB x NZW F, hybrid (B/W) mice spontaneously develop antinucleic acid antibodies and immune complex glomerulonephritis which resembles human systemic lupus erythematosus (Helyer & Howie, 1963a; Larnbert & Dixon, 1968). Cell-mediated immunity is deficient with age in B/W mice, e.g. the spleen cells of old mice lack the ability to induce a graft-vs-host reaction (Gerber, Hardin & others, 1974), and to respond to mitogens (Leventhal & Talal, 1970). and the rejection process of these mice against skin allografts (Gelfand & Steinberg, 1973) and some tumors (Gazdar, Beitzel & Talal, 1971), is impaired. Evidence has accumulated suggesting that the deficiency of suppressor T-cell activity allows the formation of autoantibody and the development of autoimmune diseases in NZB and B/W mice (Allison, Denman & Barnes, 1971; Chused, Steinberg & Parker, 1973; Barthold, Kysela & Steinberg, 1974; Steinberg & Talal, 1975: Krakauer, Waldmann & Strober, 1976; Talal & Steinberg, 1976). In addition, neonatal thymectomy results in a high incidence of disorders in B/W mice (Helyer & Howie, 1963b). Gershwin & Steinberg (1975a) reported that biweekly injections of thymocytes from young NZB mice, which include suppressor T lymphocytes, inhibited the pathogenesis of autoimmune haemolytic anaemia in syngeneic mice. Very recently a similar prophylactic effect was observed in B/W mice treated with the soluble factor from suppressor T lymphocytes (Krakauer, Strober & others, 1977). Further, it has been reported that injections of concanavalin A (Gershwin & Steinberg, 1975b) or thymosin (Gershwin, Ahmed & others, 1974; Talal & Steinberg, 1976) depressed immunologic abnormality and delayed the onset of, or prevented development of, autoimmune diseases in NZB or B/W mice. From the above evidence, we considered that agents activating the T-cell function might prevent the appearance of autoantibody and the development of autoimmune desease in B/W mice.

Granulocyte colony-stimulating factor exacerbates the acute lung injury and pulmonary fibrosis induced by intratracheal administration of bleomycin in rats

We investigated the effects of granulocyte colony-stimulating factor (G-CSF) on lung injury induced by intratracheal administration of bleomycin (BLM, 2 mg/200 micro1) in rats. In experiment 1, G-CSF (10, 30 and 100 microg/kg/day, s.c.) was administered to rats treated with BLM or saline for 7 days starting immediately after BLM administration. In rats receiving G-CSF alone, a large number of neutrophils were noted in the pulmonary capillaries, although there were no lung lesions. In rats receiving BLM alone, diffuse alveolar damage was observed. The administration of G-CSF to BLM-treated rats increased the total lung lesion per unit of pulmonary parenchyma (total lung lesion %) along with increases in the peripheral neutrophil count and the number of neutrophils infiltrating in the pulmonary lesion in a dose-dependent fashion. In experiment 2, 100 microg/kg/day of G-CSF was administered to rats treated with BLM or saline for up to 28 days starting immediately after BLM administration. The administration of 100 microg/kg/day of G-CSF to BLM-treated rats showed no effects at 14 days but it increased the lung lesion % and the score of lung fibrosis along with the increase in the number of neutrophils infiltrating in the pulmonary lesion at 28 days. These findings suggest that G-CSF administration to BLM-treated rats influenced and exacerbated the BLM-induced acute lung injury, and also exacerbated pulmonary fibrosis in a dose-dependent fashion. The exacerbation of lung injury coincided with the marked increase in the peripheral neutrophil count and the number of neutrophils infiltrating in the pulmonary lesion.

Effects of a synthetic progestogen, altrenogest, on oestrus synchronisation and fertility in miniature pigs

Groups of six, six and eight miniature gilts, respectively, received 5, 10 or 15 mg/day of altrenogest for 18 days, and the numbers of corpora lutea and residual follicles were counted approximately 14 days after the treatment by an exploratory laparotomy. They were compared with the numbers in a control group of eight gilts which were examined six to eight days after oestrus. The interval between the last dose of altrenogest and the onset of oestrus increased with the dose of altrenogest, and was significantly longer after the treatments with 10 or 15 mg/day than after 5 mg/day (P<0.01). Significantly more corpora lutea were observed in the gilts receiving 5 or 10 mg/day of altrenogest than in the control gilts (P<0.01). Groups of six, seven and six miniature gilts that had respectively received 5, 10 or 15 mg/day of altrenogest were artificially inseminated; four, six and five of the gilts in these groups farrowed, and their mean (sd) litter sizes were 5.5 (2.4), 6.8 (1.2) and 5.0 (2.3), respectively. All six of a group of control gilts farrowed and their mean litter size was 5.8 (1.2).