Production of porcine cloned embryos derived from cells conditionally expressing an exogenous gene using Cre-loxP

SummaryIt is increasingly evident that conditional gene expression in pigs is necessary to make transgenic models. In this study, we investigated conditional expression in porcine fetal fibroblasts using Cre-loxP recombination, a system that has had limited application in large animals to date. Transformed fibroblasts were reprogrammed in enucleated oocytes to support further early embryonic development. Fetal fibroblasts from miniature pigs were used for transfection with a plasmid that contained a red fluorescent protein marker (pCALNL-DsRed) and a floxed neomycin-resistance gene. Cells were selected with 750 μg/ml neomycin for 2 weeks following transfection but did not express DsRed after visualization under a fluorescence microscope. Expression was achieved only after transient transfection with plasmid DNA that expressed the Cre recombinase enzyme. The cells that expressed DsRed were used for somatic cell nuclear transfer (SCNT). A total of 121 oocytes were used for SCNT and 76 cloned embryos (62.8%) were seen to have cleaved. Six blastocysts developed after SCNT and expressed DsRed. Deletion of the floxed neomycin-resistance gene was confirmed by reverse transcription polymerase chain reaction (RT-PCR) in cloned blastocysts. This study demonstrated that Cre-loxP recombination can be conducted successfully in miniature pig fibroblasts and that the sequentially transformed cells can develop to the pre-implantation embryo stage via SCNT.

A Bicistronic Subgenomic mRNA Encodes both the ORF2 and ORF3 Proteins of Hepatitis E Virus

ABSTRACT Hepatitis E virus replicons containing the neomycin resistance gene expressed from open reading frames (ORFs) 2 and 3 were transfected into Huh-7 cells, and stable cell lines containing functional replicons were selected by constant exposure to G418 sulfate. Northern blot analyses detected full-length replicon RNA and a single subgenomic RNA. This subgenomic RNA, which was capped, initiated at nucleotide 5122 downstream of the first two methionine codons in ORF3 and was bicistronic; two closely spaced methionine codons in different reading frames were used for the initiation of ORF3 and ORF2 translation.

Genetic Evidence That an 11 Amino Acid β-Hairpin Loop in the Cytoplasmic Domain of Band 3 Is Responsible for at Least 50% of Ankyrin Binding in Mouse Erythrocytes.

The anion exchange protein (band 3) is an integral erythrocyte membrane protein that is associated with the spectrin-actin cytoskeleton through ankyrin, which binds to both band 3 and spectrin. Improper band 3/ankyrin interactions result in erythrocyte instability leading to spherocytosis and hemolytic anemia. The crystal structures of ankyrin and band 3 predict that the ankyrin amino-terminus, containing comb-like ANKYRIN repeat subdomains, binds to a β-hairpin loop (residues 175-185) in the band 3 cytoplasmic domain. We have previously shown that replacement of human band 3 residues 175–185 with a di-glycine bridge eliminates the binding of an ankyrin fragment containing domains 3 and 4 to the cytoplasmic domain of band 3 in vitro. To test this model for ankyrin/band 3 interactions in vivo, we used homologous recombination in embryonic stem cells to replace the corresponding mouse band 3 sequences, residues 189–199 in exon 7, with a di-glycine bridge to create a knock-in transgenic mouse. Analysis of mRNA expression in transgenic mice carrying the targeted locus demonstrated that the level of mRNA from the mutant allele (which contains the neomycin resistance gene used for positive selection of embryonic stem cells) was 50-70 fold lower than the level of mRNA from the wild type allele. Heterozygous transgenic mice were mated to mice expressing the Cre recombinase protein in the germ line to remove the neomycin resistance gene. After removal of the neomycin resistance gene from the band 3 locus, mice homozygous for the 189–199 deletion/di-glycine insertion were born in a normal Mendelian ratio and had normal numbers of red blood cells, white blood cells and platelets. Quantitative RT-PCR analysis showed that the level of band 3 mRNA from the wild type and mutant alleles were identical. SDS poly acrylamide gel electrophoresis and Western Blot analysis demonstrated normal levels of band 3 and other red cell membrane proteins in wild type, heterozygous and homozygous mutant erythrocytes. Homozygous mutant mice showed a modest but significant increase in osmotic fragility (p<0.01) compared to wild type or heterozygous mice. Ankyrin binding to inside out vesicles (IOV) from wild type, heterozygous and homozygous mutant erythrocytes was measured using an 125I labeled peptide containing domains 3 and 4 of mouse ankyrin. Ankyrin binding to homozygous mutant IOVs was 50% or less than the level of ankyrin binding to WT IOVs. We conclude from these studies that the 11 amino acid β-hairpin loop encoded by residues 189–199 of the band 3 gene is responsible for at least 50% of the ankyrin binding in mouse erythrocytes. We predict that mutations in this region of human band 3 would lead to a phenotype of Hereditary Spherocytosis similar to that seen with ankyrin deficiency. Space filling models of the cytoplasmic domain of band 3 have identified a second homologous β-hairpin loop encoded by residues 63-75, which is the target of our future investigation.

Autotransplantation in mdx Mice of mdx Myoblasts Genetically Corrected by an HSV-1 Amplicon Vector

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder, characterized by a lack of dystrophin. To eliminate the need for immunosuppressive drugs, transplantation of genetically modified autologous myoblasts has been proposed as a possible therapy for this myopathy. An HSV-1 amplicon vector (HSVDGN), containing a 17.3-kb full-length MCK-driven mouse dystrophin cDNA, an eGFP gene, and a neomycin resistance gene driven by CMV or SV40 promoters, respectively, was constructed and used to transduce mdx primary myoblasts. The presence of the eGFP and neomycin resistance genes facilitated the evaluation of the initial transduction efficiency and the permanent transduction frequency. At low multiplicities of infection (MOI 1–5), the majority of myoblasts (60–90%) expressed GFP. The GFP-positive mdx myoblasts were sorted by FACS and selected with neomycin (300 μg/ml) for 2 weeks. Up to 2% of initially infected mdx myoblasts stably expressed the three transgenes without further selection at that time. These altered cells were grafted into the tibialis anterior muscles of 18 mdx mice. Some of the mice were immunosuppressed with FK506 due to the anticipation that eGFP and the product of neomycin resistance gene might be immunogenic. One month after transplantation, numerous muscle fibers expressing mouse dystrophin were detected by immunohistochemistry, in both immunosuppressed (10–50%) and nonimmunosuppressed (5–25%) mdx mice. Our results demonstrated the capability of permanently expressing a full-length dystrophin in dystrophic myoblasts with HSV-1 amplicon vector and raised the possibility of an eventual treatment of DMD based on the transplantation of genetically modified autologous myoblasts.

Use of Allogeneic Bone Marrow Labeled with Neomycin Resistance Gene to Examine Bone Marrow-Derived Chimerism in Experimental Organ Transplantation

Posttransplant infusion of viable donor bone marrow cells (DBMC) has been shown in our previous studies to promote acceptance of incompatible kidney allografts in rhesus monkeys after treatment with polyclonal antithymocyte globulin to deplete peripheral T-lymphocytes. In this nonhuman primate model, the infusion of the DBMC is requisite for the induction of functional graft tolerance and specific MLR and CTLp unresponsiveness, although the relevant role and fate of bone marrow-derived chimeric cells is uncertain. Standard immunological and molecular techniques applied to this monkey model are unable to differentiate between chimeric cells derived from the infused DBMC and those derived from allograft-borne passenger leukocyte emigrants. To distinguish chimerism due to infused DBMC, we transduced DBMC with a functional neomycin resistance gene (Neor) using the retroviral vector pHSG-Neo. Neor-Mransduced BMC were infused into recipients approximately 2 wk after kidney transplantation and treatment with rabbit antithymocyte globulin. No maintenance immunosuppressive drugs were given. Genomic DNA isolated from peripheral blood leukocytes was used to monitor the presence of Neor-positive cells. Tissue samples obtained at necropsy also were assessed for Neor-positive chimeric cells. The presence of DBMC-derived chimerism was assessed by polymerase chain reaction using Neor sequence-specific primers (PCR-SSP). Chimerism was detectable in recipient tissues at various times for up to 6 mo after DBMC infusion. These studies using gene transduction methodology indicate that a stable genetic marker can provide capability to examine DBMC-derived chimerism for prolonged periods in a nonhuman primate model. This approach should facilitate future studies in preclinical models to study the role and type of chimeric cell lineages in relation to functional allograft tolerance.