ABSTRACT
OBJECTIVE: Aggregation of human amylin/islet amyloid polypeptide (hA/hIAPP) into small soluble beta-sheet-containing oligomers is linked to islet beta-cell degeneration and the pathogenesis of type 2 diabetes. Here, we used tetracycline, which modifies hA/hIAPP oligomerization, to probe mechanisms whereby hA/hIAPP causes diabetes in hemizygous hA/hIAPP-transgenic mice. RESEARCH DESIGN AND METHODS: We chronically treated hemizygous hA/hIAPP transgenic mice with oral tetracycline to determine its effects on rates of diabetes initiation, progression, and survival. RESULTS: Homozygous mice developed severe spontaneous diabetes due to islet beta-cell loss. Hemizygous transgenic animals also developed spontaneous diabetes, although severity was less and progression rates slower. Pathogenesis was characterized by initial islet beta-cell dysfunction followed by progressive beta-cell loss. Islet amyloid was absent from hemizygous animals with early-onset diabetes and correlated positively with longevity. Some long-lived nondiabetic hemizygous animals also had large islet-amyloid areas, showing that amyloid itself was not intrinsically cytotoxic. Administration of tetracycline dose-dependently ameliorated hyperglycemia and polydipsia, delayed rates of diabetes initiation and progression, and increased longevity compared with water-treated controls. CONCLUSIONS: This is the first report to show that treating hA/hIAPP transgenic mice with a modifier of hA/hIAPP misfolding can ameliorate their diabetic phenotype. Fibrillar amyloid was neither necessary nor sufficient to cause diabetes and indeed was positively correlated with longevity therein, whereas early- to mid-stage diabetes was associated with islet beta-cell dysfunction followed by beta-cell loss. Interventions capable of suppressing misfolding in soluble hA/hIAPP oligomers rather than mature fibrils may have potential for treating or preventing type 2 diabetes.
Subject(s)
Amyloid/genetics , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/prevention & control , Protein Synthesis Inhibitors/therapeutic use , Tetracycline/therapeutic use , Administration, Oral , Animals , Blood Glucose/drug effects , Blood Glucose/metabolism , Blotting, Northern , DNA Primers , Diabetes Mellitus, Type 2/blood , Disease Progression , Glucose Tolerance Test , Homozygote , Humans , Insulin-Secreting Cells/pathology , Islet Amyloid Polypeptide , Islets of Langerhans/pathology , Mice , Mice, Transgenic , Polymerase Chain Reaction , Protein Synthesis Inhibitors/administration & dosage , Tetracycline/administration & dosageABSTRACT
The present study investigated whether protamine sulfate can be used to improve the efficiency of bovine sperm mediated transgenesis (SMT) by protecting the plasmid pCX-EGFP against nuclease activity. A high proportion (31%) of bovine spermatozoa transfected with the plasmid pCX-EGFP maintain their motility. Using an in vitro assay, protamine sulfate protected the plasmid against degradation by DNase I. However, upon transfecting spermatozoa, the plasmid remained intact regardless of whether it was complexed to protamine sulfate. When in vitro fertilisation (IVF) was undertaken using transfected sperm, 14.6 and 10.2% of blastocysts derived from pCX-EGFP only and pCX-EGFP-protamine transfected sperm, respectively, were PCR positive for the plasmid. In conclusion, using spermatozoa transfected with either pCX-EGFP or pCX-EGFP-protamine complexes, produced PCR positive blastocysts after SMT. However, the use of protamine sulfate does not improve the efficiency of SMT suggesting that factors other than nuclease activity could be limiting.
Subject(s)
Cattle/physiology , DNA/metabolism , Deoxyribonucleases/antagonists & inhibitors , Plasmids/genetics , Protamines/pharmacology , Transfection/veterinary , Animals , Animals, Genetically Modified , Cattle/genetics , DNA/drug effects , DNA/genetics , Deoxyribonucleases/metabolism , Embryo Culture Techniques/veterinary , Fertilization in Vitro/veterinary , Fluorescent Dyes , Gene Transfer Techniques/veterinary , Male , Plasmids/drug effects , Plasmids/metabolism , Polymerase Chain Reaction/methods , Polymerase Chain Reaction/veterinary , Sperm Injections, Intracytoplasmic/veterinary , Sperm Motility , Spermatozoa , Transfection/methodsABSTRACT
The extraembryonic ectoderm (ExE) is essential for mammalian placental formation and survival of the embryo in utero. We have obtained a mouse model lacking the ExE, by targeted deletion of the transcription factor Elf5. Although Elf5 mutant embryos implant and form an ectoplacental cone, no trophoblast stem (TS) cells can be derived, indicating that the absence of ExE is a result of the lack of TS cell maintenance. Embryos without ExE tissue are able to form the anterior visceral endoderm but fail to undergo gastrulation, demonstrating an essential role for the ExE in embryonic patterning during a defined window of development.
Subject(s)
Body Patterning/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Ectoderm/physiology , Gastrula/physiology , Mice/embryology , Transcription Factors/genetics , Transcription Factors/metabolism , Trophoblasts/cytology , Animals , Blotting, Southern , DNA Primers , Gene Deletion , Genotype , In Situ Hybridization , Mutation/genetics , Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
To enhance milk composition and milk processing efficiency by increasing the casein concentration in milk, we have introduced additional copies of the genes encoding bovine beta- and kappa-casein (CSN2 and CSN3, respectively) into female bovine fibroblasts. Nuclear transfer with four independent donor cell lines resulted in the production of 11 transgenic calves. The analysis of hormonally induced milk showed substantial expression and secretion of the transgene-derived caseins into milk. Nine cows, representing two high-expressing lines, produced milk with an 8-20% increase in beta-casein, a twofold increase in kappa-casein levels, and a markedly altered kappa-casein to total casein ratio. These results show that it is feasible to substantially alter a major component of milk in high producing dairy cows by a transgenic approach and thus to improve the functional properties of dairy milk.
