Use of nonautologous microencapsulated fibroblasts in growth hormone gene therapy to improve growth of midget swine

The aim of the present study was to investigate the expression activity, both in vitro and in vivo, of the porcine growth hormone complementary DNA (pGH cDNA) in porcine fetal fibroblast (PFF) cells, The pGH gene had been constructed inside the bicistronic retroviral vector PSN and subsequently transfected into PFF cells further encapsulated with immunoprotective microcapsules, This would provide a way to evaluate the improvement in growth performance of Tao-Yuan swine by the use of nonautologous microencapsulated fibroblasts carrying the pGH cDNA via the technique of somatic gene therapy. Results from Southern blot analysis confirmed that the full length of the pGH cDNA was completely integrated into the genome of the PFF cells after they had been infected one to four times using a PSN retroviral vector. Moreover, Northern blot analysis showed that high transcription activity was present in clones infected twice, and exogenous pGH secretion was found when the pGH-infected PFF had been further cultured for 48 hr in vitro and subjected to immunoblot assay. Encapsulation of the pGH-PFF with an alginate-poly-L-lysine-alginate membrane did not show any deterioration in their proliferation and survival both in vitro and in vivo. The pGH gene in encapsulated recombinant fibroblasts was fully expressed after it had been transplanted into the peritoneal cavity of the Tao-Yuan swine, and reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed on the microcapsules retrieved 1 month later. The feasibility of pGH gene therapy to improve midget Tao-Yuan swine growth enhancement is further supported by the fact that transplantation of the encapsulated recombinant fibroblast cells resulted in a much more significant increase in weight gain than in those swine in either the age-matched untreated control group or in those that had been transplanted with uncapsulated recombinant PFF cells (10.56 +/- 1.01 kg versus 6.95 +/- 0.94 and 5.27 +/- 1.30 kg; p < 0.05), These experimental data suggest that growth hormone gene therapy did provide an alternative approach for growth improvement in midget Tao-Yuan swine

Granzyme G is expressed in the two-cell stage mouse embryo and is required for the maternal-zygotic transition

Background: Detailed knowledge of the molecular and cellular mechanisms that direct spatial and temporal gene expression in pre-implantation embryos is critical for understanding the control of the maternal-zygotic transition and cell differentiation in early embryonic development. In this study, twenty-three clones, expressed at different stages of early mouse development, were identified using differential display reverse transcription polymerase chain reaction (DDRT-PCR). One of these clones, which is expressed in 2-cell stage embryos at 48 hr post-hCG injection, shows a perfect sequence homology to the gene encoding the granzyme G protein. The granzyme family members are serine proteases that are present in the secretory granules of cytolytic T lymphocytes. However, the pattern of granzyme G expression and its function in early mouse embryos are entirely unknown. Results: Upon the introduction of an antisense morpholino (2 mM) against granzyme G to knock-down endogenous gene function, all embryos were arrested at the 2- to 4-cell stages of egg cleavage, and the de novo synthesis of zygotic RNAs was decreased. The embryonic survival rate was dramatically decreased at the late 2-cell stage when serine protease-specific inhibitors, 0.1 mM 3,4-dichloroisocoumarin (3,4-DCI), and 2 mM phenyl methanesulphonyl fluoride (PMSF), were added to the in vitro embryonic culture medium. Survival was not affected by the addition of 0.5 mM EDTA, a metalloproteinase inhibitor. Conclusion: We characterized for the first time the expression and function of granzyme G during early stage embryogenesis. Our data suggest that granzyme G is an important factor in early mouse embryonic development and may play a novel role in the elimination of maternal proteins and the triggering of zygotic gene expression during the maternal-zygotic transition

Temporal and spatial expression of biologically active human factor VIII in the milk of transgenic mice driven by mammary-specific bovine alpha-lactalbumin regulation sequences

Hemophilia A is one of the major inherited bleeding disorders caused by a deficiency or abnormality in coagulation factor VIII (FVIII). Hemophiliacs have been treated with whole plasma or purified FVIII concentrates. The risk of transmitting blood-borne viruses and the cost of highly purified FVIII are the major factors that restrict prophylaxis in hemophilia therapy. One of the challenges created by the biotechnology revolution is the development of methods for the economical production of highly purified proteins in large scales. Recent developments indicate that manipulating milk composition using transgenesis has focused mainly on the mammary gland as a bioreactor to produce pharmaceuticals. In the present study, a hybrid gene containing bovine alpha-lactalbumin and human FVIII cDNA was constructed for microinjection into the pronuclei of newly fertilized mouse eggs. The alphaLA-hFVIII hybrid gene was confirmed to be successfully integrated and stably germ-line transmitted in 12 (seven females/five males) lines. Western-blot analysis of milk samples obtained from eight of the transgenic founders and F1 offspring indicated that the recombinant hFVIII was secreted into the milk of the transgenic mice. The concentrations of rFVIII ranged from 7.0 to 50.2 mug/ml, over 35-200-fold higher than that in normal human plasma. Up to 13.4 U/ml of rFVIII was detected in an assay in which rFVIII restored normal clotting activity to FVIII-deficient human plasma

Growth hormone gene polymorphisms and growth performance traits in Duroc, Landrace and Tao-Yuan pigs

This study investigated the restriction fragment length polymorphism (RFLP) of the porcine growth hormone (pGH) gene in Duroc, Landrace, and Tao-Yuan pigs and its effects on growth performance and levels of plasma growth hormone in peripheral circulation. Genomic DNA extracted from 81 Tao-Yuan, 60 Landrace and 48 Duroc pigs were subjected to Southern blot hybridization with a pGH cDNA probe. Polymorphism was detected with the restriction enzymes TaqI and DraI. A comparison of these three breeds showed significant differences in allelic frequencies. Blood samples for radioimmunoassay (RIA) of GH were collected biweekly during the experimental period from pigs 12 to 40 weeks of age. Tao-Yuan pigs showed a mean plasma GH level (2.51 +/- 1.23 ng/mL) that was much lower than that of the Landrace (3.80 +/- 1.52 ng/mL) and Duroc (4.20 +/- 1.03 ng/mL.) pigs (P< 0.05). Moreover, the Tao-Yuan pigs also showed poorer growth performance than the Landrace and the Duroc pigs both in the daily weight gain (0.37 +/- 0.06 vs. 0.67 +/- 0.05 and 0.70 +/- 0.05 kg/day, P< 0.01) and feed efficiency (3.12 +/- 0.28 vs. 2.60 +/- 0.14 and 2.52 +/- 0.12, P< 0.05). These results suggest that the growth performance trait in these pigs is highly correlated with their growth hormone genotype. (C) 2000 by Elsevier Science Inc

Growth enhancement of fowls by dietary administration of recombinant yeast cultures containing enriched growth hormone

In present study the methylotrophic yeast, Pichia pastoris, was used to express a recombinant growth hormone (rGH) gene of swine, A synthetic secretion cassette was constructed using the promoter of the alcohol oxidase1 gene (AOX1), and a alpha-factor signal peptide. After electroporatic transformation and zeocin selection, several clones exhibited high levels of rGH protein expression constituting more than 20% of total yeast protein. Over 95% of rGH was shown to be export into the culture supernatant, Yeast transformant containing the highest recombinant growth hormone level (rGH yeast) and native GS115 Pichia pastoris (non-rGH yeast, as a control) were separately cultured, harvested and adsorbed by wheat bran, Yeast cultures of four dosages (0.05, 0.1, 0.2, and 0.4%) were mixed respectively with chick basal diet and fed to simulated country chickens for 9 weeks. The results showed that, when compared to control chicks, the percentage of body weight gain was improved significantly (P<0.05) in chicks fed with diets containing 0.1 or 0.2% rGH-rich yeast culture at brooding stage, and in chicks fed with 0.4% rGH-rich yeast culture at growing stage. The average weight gain in rGH yeast treated groups for the full-term (0 to 63d) and short term (43 to 63d) of growth were 10.6 and 9.4%, respectively, better than the non-rGH yeast control group. These experimental data suggest that the use of rGH-containing yeast as a supplement in fed provided an alternative approach for growth improvement in simulated country chickens. (C) 2000 Elsevier Science Inc. All rights reserved

Proliferin enhances microvilli formation and cell growth of neuroblastoma cells

Proliferins (also termed mitogen-regulated proteins; MRP/PLFs) belong to the prolactin gene family. Mrp/Plfs are involved in angiogenesis of the uterus and placenta and maximally expressed during midgestation and decline through the remainder of the gestation period in mouse placenta. The tissue expressions of Mrp/Plfs are mainly documented in placenta, hair follicles of skin and in wound healing. In this report, we demonstrate that Plf1, Plf1 minus exon3, Plf2 and Mrp3 but not Mrp4 are expressed in mouse whole brain by diagnostic RT-PCR and Western blotting. The expression levels of Mrp/Plf mRNAs in mouse brains were low during the neonatal period, but higher in embryonic and adult stages, indicating Mrp/Plfs expression profiles are different in mouse brain and placenta. Interestingly, endogenous Mrp/Plfs were detected using immunostaining both in mouse brain sections and the neuroblastoma cell line, Neuro-2a cells. The function of PLF1 was explored by expressing exogenous PLF1 in Neuro-2a cells. This resulted in increased microvilli. Neuro-2a cells with stable expression of PLF1 had increased proliferation compared with normal and stable expressing EGFP cells when cell reached saturation density. Together these data, strongly suggest that MRP/PLFs mediate microvilli formation and contribute to cell proliferation of neuroblastoma cells. (c) 2006 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved