A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases. Here we describe the use of somatic cell nuclear transfer to produce cloned transgenic cats that systemically express red fluorescent protein. Immature oocytes were collected from superovulating cat ovaries. Donor fibroblasts were obtained from an ear skin biopsy of a white male Turkish Angora cat, cultured for one to two passages, and subjected to transduction with a retrovirus vector designed to transfer and express the red fluorescent protein (RFP) gene. A total of 176 RFP cloned embryos were transferred into 11 surrogate mothers (mean = 16 +/- 7.5 per recipient). Three surrogate mothers were successfully impregnated (27.3%) and delivered two liveborn and one stillborn kitten at 65 to 66 days of gestation. Analysis of nine feline-specific microsatellite loci confirmed that the cloned cats were genetically identical to the donor cat. Presence of the RFP gene in the transgenic cat genome was confirmed by PCR and Southern blot analyses. Whole-body red fluorescence was detected 60 days after birth in the liveborn transgenic (TG) cat but not in the surrogate mother cat. Red fluorescence was detected in tissue samples, including hair, muscle, brain, heart, liver, kidney, spleen, bronchus, lung, stomach, intestine, tongue, and even excrement of the stillborn TG cat. These results suggest that this nuclear transfer procedure using genetically modified somatic cells could be useful for the efficient production of transgenic cats.
We successfully produced second-generation cloned cats by somatic cell nuclear transfer (SCNT) using skin cells from a cloned cat. Skin cells from an odd-eyed, all-white male cat (G0 donor cat) were used to generate a cloned cat (G1 cloned cat). At 6 months of age, skin cells from the G1 cloned cat were used for SCNT to produce second-generation cloned cats. We compared the in vitro and in vivo development of SCNT embryos that were derived from the G0 donor and G1 cloned donor cat's skin fibroblasts. The nuclei from the G0 donor and G1 cloned donor cat's skin fibroblasts fused with enucleated oocytes with equal rates of fusion (60.7% vs. 58.8%, respectively) and cleavage (66.3% vs. 63.4%). The 2-4-cell SCNT embryos were then transferred into recipients. One of the five recipients of G0 donor derived NT embryos (20%) delivered one live male cloned kitten, whereas 4 of 15 recipients of the G1 cloned donor cat derived NT embryos (26%) delivered a total of seven male second-generation cloned kittens (four live kittens from one surrogate, plus two stillborn kittens, and one live kitten that died 2d after birth from three other surrogate mothers). The four second-generation cloned kittens from the same surrogate all had a white coat color; three of the four second-generation cloned kittens had two blue eyes, and one of the second-generation cloned kittens had an odd-eye color. Despite low cloning efficiency, cloned cats can be used as donor cats to produce second-generation cloned cats.
Mesenchymal stem cells (MSCs) secrete a variety of neuroregulatory molecules, such as nerve growth factor, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor, which upregulate tyrosine hydroxylase (TH) gene expression in PC12 cells. Enhancing TH gene expression is a critical step for treatment of Parkinson's disease (PD). The objective of this study was to assess the effects of co-culturing PC12 cells with MSCs from feline bone marrow on TH protein expression. We divided the study into three groups: an MSC group, a PC12 cell group, and the combined MSC + PC12 cell group (the co-culture group). All cells were cultured in DMEM-HG medium supplemented with 10% fetal bovine serum for three days. Thereafter, the cells were examined using western blot analysis and immunocytochemistry. In western blots, the co-culture group demonstrated a stronger signal at 60 kDa than the PC12 cell group (p<0.001). TH was not expressed in the MSC group, either in western blot or immunocytochemistry. Thus, the MSCs of feline bone marrow can up-regulate TH expression in PC12 cells. This implies a new role for MSCs in the neurodegenerative disease process.
Production of transgenic animals is highly desirable for biotechnology and basic research. Therefore, a method of producing genetically modified cats through genetic engineering is important for generating biomedical models of human diseases. We investigate reproductive ability of red fluorescence protein (RFP) transgenic cloned male cat (RFP TG cat) in natural mating with domestic female cat. One domestic female cat in natural mating with RFP TG cat delivered 6 kittens of which 3 (2 female and 1 male) showed RFP expression of parental line. Among the 3 RFP expressing kittens, 1 died at day 5 and were subjected to PCR analysis for confirmation of RFP gene expression. Red fluorescence protein gene was detected in tissue samples including liver, muscle, brain, large intestine, bladder, spleen, sexual organ, small intestine, kidney, heart, stomach, pancreas, lungs, and skin of the kitten. These results indicated that RFP transgenic cloned male cat have normal reproductive fertility, and the stability of transmission of the RFP transgene through the germ line and its normal expression in offspring. Supported by KOSEF (M10525010001-05N2501-00110). S. J. Cho, Y. S. Lee, E. G. Choi, and J. I. Bang were supported by a scholarship from the Post BK21 Program, the Ministry of Education, Science and Technology, Korea.
Context Although vitrification is commonly used for oocyte cryopreservation, the cryogenic damage results in poor developmental capacity of oocytes after freezing. Nano-cryopreservation is one of the new methods of vitrification developed in recent years. However, the effect of nano-cryopreservation on mature bovine oocytes remains to be elucidated. Aims This study aimed to verify the effect of using hydroxyapatite (HA) nanoparticles (NPs) on the vitrification of bovine metaphase II (MII)-stage oocytes. Methods Bovine MII-stage oocytes were exposed to different HA concentrations (0.01%, 0.05%, and 0.10%) in vitrification solution (VS). After IVF (in vitro fertilisation) and IVC (in vitro culture), the toxicity of HA was assessed by cleavage and blastocyst rates. A suitable concentration of HA nanoparticles was selected according to the results of the first experiment. and then vitrification-thawing was measured. The effect of HA on the developmental capacity of oocytes was assessed by oocyte cleavage rate and blastocyst rate. The mitochondrial membrane potential (MMP) and the intracellular reactive oxygen levels (ROS) of oocytes were measured by staining with a fluorescence probe (JC-1) and an ROS kit after nano-cryopreservation. Key results The addition of 0.05% HA to the VS did not affect the oocyte morphology; the proportion of oocytes developing with normal morphology was 96.72%. In contrast, this proportion it significantly decreased at a concentration of 0.1% HA in VS (91.69%; P < 0.05). The cleavage rates (56.95% vs 51.20%, 50.67%; P < 0.05) of bovine oocytes exposed to 0.05% HA were significantly higher than in the VS group and VS + 0.1% HA group. After the vitrification-thawing, the oocyte cleavage rates (41.07% vs 33.97%; P < 0.05) and blastocyst rates (12.35% vs 7.38%; P < 0.05) were significantly higher in the VS + 0.05% HA group compared to the VS group. At the 0.05% HA concentration, nano-cryopreserved oocytes had significantly higher MMP (1.35 ± 0.24) and significantly lower ROS (1.43 ± 0.05) than the VS group (P < 0.05, mean ± s.e.m.). Conclusions The addition of 0.05% HA-NPs in VS could promote bovine MII-stage oocytes’ developmental ability after vitrification. This effect may be caused partly by increased mitochondrial membrane activity and decreased ROS. Implications HA-NPs may be a new class of cryoprotective agent suitable as components for oocyte vitrification.
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