Culture systems promote development at rates lower than the in vivo environment. Here, we evaluated the embryo’s transcriptome to determine what the embryo needs during development. A previous mRNA sequencing endeavor found SLC7A1, an arginine transporter, to be up-regulated in in vitro compared to in vivo cultured embryos. We added different concentrations of arginine to our culture medium to meet the needs of the embryo. Increasing arginine from 0.12 to 1.69 mM improved the number of embryos that developed to the blastocyst stage. These blastocysts also had more total nuclei compared to controls and specifically, more trophectoderm nuclei. Embryos cultured in 1.69 mM arginine had lower SLC7A1 levels and had higher abundance of messages involved with glycolysis (HK1, HK2, and GPT2) and decreased expression of genes involved with blocking the TCA cycle (PDK1) as well as the pentose phosphate pathway (TALDO1). PRMT1, PRMT3 and PRMT5 expression throughout development was not affected by arginine. DDAH1 and DDAH2 message was found to be differentially regulated through development and DDAH2 protein was localized to the nuclei of blastocysts. Arginine has a positive effect on preimplantation development and may be affecting the NO-DDAH-PRMT axis.

Myostatin (MSTN) is a well-known negative regulator of muscle growth. Animals that possess mutations within this gene display an enhanced muscling phenotype, a desirable agricultural trait. Increased neonatal morbidity is common, however, resulting from complications arising from the birth of offspring with increased fetal muscle mass. The objective of the current research was to generate an attenuated MSTN-null phenotype in a large-animal model using RNA interference to enhance muscle development without the detrimental consequences of an inactivating mutation. To this end, we identified a series of short interfering RNAs that demonstrated effective suppression of MSTN mRNA and protein levels. To produce transgenic offspring capable of stable MSTN suppression in vivo, a recombinant lentiviral vector expressing a short hairpin RNA (shRNA) targeting MSTN for silencing was introduced into bovine fetal fibroblasts. These cells were used as nucleus donors for somatic cell nuclear transfer (SCNT). Twenty blastocysts were transferred into seven recipient cows resulting in five pregnancies. One transgenic calf developed to term, but died following delivery by Caesarean-section. As an alternative strategy, microinjection of recombinant lentiviral particles into the perivitelline space of in vitro-produced bovine zygotes was utilized to produce 40 transgenic blastocysts that were transferred into 14 recipient cows, resulting in 7 pregnancies. Five transgenic calves were produced, of which three expressed the transgene. This is the first report of transgenic livestock produced by direct injection of a recombinant lentivirus, and expressing transgenes encoding shRNAs targeting an endogenous gene (myostatin) for silencing.

Pantoea stewartii subsp. stewartii is a bacterial pathogen of corn. Its pathogenicity depends on the translocation of effector proteins into host cells by the Hrp type III secretion system. We previously showed by genetic analysis that the HrpX sensor kinase and the HrpY response regulator are at the head of a complex cascade of regulators controlling hrp/hrc secretion and wts effector genes. This cascade also includes the HrpS response regulator and the HrpL alternative sigma factor. These regulators are shared among many important plant pathogens in the genera Pantoea, Erwinia, and Pseudomonas. In this study, we dissect the regulatory elements in the hrpS promoter region, using genetic and biochemical approaches, and show how it integrates various environmental signals, only some of which are dependent on phosphorylation of HrpY. Primer extension located the transcriptional start site of hrpS at a 70 promoter 601 bp upstream of the open reading frame. Electrophoretic mobility shift assays and DNase I footprinting analysis demonstrated that HrpY binds to conserved regulatory elements immediately adjacent to this promoter, and its binding affinity was increased by phosphorylation at D57. A consensus sequence for the two direct repeats bound by HrpY is proposed. Deletion analysis of the promoter region revealed that both the HrpY binding site and additional sequences farther upstream, including a putative integration host factor binding site, are required for hrpS expression. This finding suggests that other unknown regulatory proteins may act cooperatively with HrpY.The hrp/hrc genes of gram-negative, phytopathogenic bacteria encode type III secretion systems that deliver effector proteins into host cells. These proteins may elicit cell death or suppress host defenses. They are required for infection and colonization of plants by all biotrophic pathogens of the genera Pseudomonas, Ralstonia, Xanthomonas, Erwinia, and Pantoea (3). They also play a role in rapid establishment of necrotrophic, soft-rot bacteria in the genus Erwinia, where enzymatic tissue disintegration is delayed by hrp mutations (33). Pantoea stewartii subsp. stewartii (synonym, Erwinia stewartii) causes vascular wilting and leaf blight of sweet corn and maize. This pathogen carries a group I hrp gene cluster (1,7,8,11), similar to those in Pseudomonas, Erwinia, and other Pantoea species. The group I hrp gene clusters are regulated by the HrpS enhancer-binding protein and the HrpL extracytoplasmic function alternative sigma factor (12, 25, 36-39). P. stewartii hrp genes are also controlled by the HrpX/HrpY two-component regulatory system, which is also present in Pantoea agglomerans pv. gypsophilae, Erwinia amylovora, Erwinia carotovora subsp. carotovora, and Erwinia chrysanthemi (19,25,27,35,39). In erwinias and pantoeas, the hrpL-hrpXY-hrpS genes have the same map order, and the corresponding proteins share greater than 80% similarity at the amino acid level (25). However, in P. stewartii, a large remnant of an insertion sequence (IS) element i...

Immune-privileged Sertoli cells (SCs) exhibit long-term survival after allotransplantation or xenotransplantation, suggesting they can be used as a vehicle for cell-based gene therapy. Previously, we demonstrated that SCs engineered to secrete insulin by using an adenoviral vector normalized blood glucose levels in diabetic mice. However, the expression of insulin was transient, and the use of immunocompromised mice did not address the question of whether SCs can stably express insulin in immunocompetent animals. Thus, the objective of the current study was to use a lentiviral vector to achieve stable expression of insulin in SCs and test the ability of these cells to survive after allotransplantation. A mouse SC line transduced with a recombinant lentiviral vector containing furin-modified human proinsulin cDNA (MSC-EhI-Zs) maintained stable insulin expression in vitro. Allotransplantation of MSC-EhI-Zs cells into diabetic BALB/c mice demonstrated 88% and 75% graft survival rates at 20 and 50 days post-transplantation, respectively. Transplanted MSC-EhI-Zs cells continued to produce insulin mRNA throughout the study (i.e., 50 days); however, insulin protein was detected only in patches of cells within the grafts. Consistent with low insulin protein detection, there was no significant change in blood glucose levels in the transplant recipients. Nevertheless, MSC-EhI-Zs cells isolated from the grafts continued to express insulin protein in culture. Collectively, this demonstrates that MSC-EhI-Zs cells stably expressed insulin and survived allotransplantation without immunosuppression. This further strengthens the use of SCs as targets for cell-based gene therapy for the treatment of numerous chronic diseases, especially those that require basal protein expression.

Although improving, the efficiency of producing offspring by somatic cell nuclear transfer (SCNT) is still low (<1.5%). Our laboratory has demonstrated that histone deacetylase inhibitor (Scriptaid) treatment of reconstructed embryos enhances blastocyst formation and cloning efficiency in pigs. It has also been shown that proteasomal inhibitor MG132 treatment for 2 h after activation of oocytes increases blastocyst rate and pregnancy rate. The current experiment was carried out to determine the effects of combined MG132 and Scriptaid treatment on early embryo development in vitro and on term development in vivo. Immediately after electrofusion and activation, SCNT oocytes were treated with 0, 1, or 10 μM MG132 for 2 h in the presence of 500 nM Scriptaid, washed and treated with Scriptaid for an additional 14 to 15 h, then cultured in porcine zygote medium 3 (PZM3) until day 6. There was no difference in percent cleavage (58.1 ± 7.2%, 62.7 ± 7.2%, and 62.5 ± 7.2%) on day 2, or total cell number (23.1 ± 2.2, 24.0 ± 2.0, and 24.5 ± 2.3 for the 0, 1, and 10 μM MG132 groups, respectively) on day 6 among the three groups. Interestingly, there was no difference in percentage of blastocysts between the 0 (18.5±4.7%) and 1 (25.1 ± 4.7%) μM MG132 treatment groups; however, compared with the 10 μM MG132 group (14.0 ± 4.7%), more embryos from the 1 μM MG132 group developed into blastocysts (p<0.05). To determine the effects on term development in vivo, two MG132 groups were included (0 and 1 μM MG132), and embryos were treated as above and transferred into synchronized surrogates after treatment. There was no difference in the oocyte-donor cell fusion rate, number of embryos transferred, pregnancy rate at days 28, 60, and at term, pigs delivered per embryo transfer, litter size, body weight at birth, nor cloning efficiency between the Scriptaid-alone control and MG132+Scriptaid combined groups. In summary, the combined treatment of MG132 and Scriptaid did not improve term development compared to Scriptaid treatment alone.