Multiple genetic modifications in pigs can essentially benefit research on agriculture, human disease and xenotransplantation. Most multi-transgenic pigs have been produced by complex and time-consuming breeding programs using multiple single-transgenic pigs. This study explored the feasibility of producing multi-transgenic pigs using the viral 2A peptide in the light of previous research indicating that it can be utilized for multi-gene transfer in gene therapy and somatic cell reprogramming. A 2A peptide-based double-promoter expression vector that mediated the expression of four fluorescent proteins was constructed and transfected into primary porcine fetal fibroblasts. Cell colonies (54.3%) formed under G418 selection co-expressed the four fluorescent proteins at uniformly high levels. The reconstructed embryos, which were obtained by somatic cell nuclear transfer and confirmed to express the four fluorescent proteins evenly, were transplanted into seven recipient gilts. Eleven piglets were delivered by two gilts, and seven of them co-expressed the four fluorescent proteins at equivalently high levels in various tissues. The fluorescence intensities were directly observed at the nose, hoof and tongue using goggles. The results suggest that the strategy of combining the 2A peptide and double promoters efficiently mediates the co-expression of the four fluorescent proteins in pigs and is hence a promising methodology to generate multi-transgenic pigs by a single nuclear transfer.

Animal models have been used to study aging for decades. In numerous aging studies, beagles are the most commonly used breed of dog. However, few studies have compared between naturally aging models and experimentally induced aging models in beagle dogs. In the present study, a D-galactose induced aging model was compared with a naturally aging model, and young adult dogs were considered as the young control group. The level of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in serum, and brain tissue were measured. Histopathological comparisons of the liver, kidneys, heart, lungs and spleen were evaluated using hematoxylin and eosin (H&E) staining, in addition, the brain was evaluated by H&E staining, and Nissl staining. The expression levels of aging-associated factors in the hippocampus, including proliferating cell nuclear antigen (PCNA), P16 and P21 were also determined through reverse transcription quantitative-polymerase chain reaction, and western blot analysis. The results indicated that D-galactose induced aging significantly increased the MDA level, while the levels of SOD and GSH-Px were diminished when compared with the young control group, which was similar to the naturally aging group. Parallel histopathological features were observed in the D-galactose induced aging and naturally aging groups compared with the young control group. However, a reduced expression level of PCNA, and increased expression levels of P16 and P21 were observed in the naturally ageing and induced aging groups compared with the young control group. The results of the current study demonstrated that the beagle dogs in D-galactose induced aging model exhibited significant similarities with the naturally aging model, providing evidence to support that the D-galactose induced aging model may be applied to aging studies.

C-C chemokine receptor type 5 (CCR5) is a major co-receptor for the entry of human immunodeficiency virus type-1 (HIV-1) into target cells. Human hematopoietic stem cells (hHSCs) with naturally occurring CCR5 deletions (Δ32) or artificially disrupted CCR5 have shown potential for curing acquired immunodeficiency syndrome (AIDS). However, Δ32 donors are scarce, heterologous bone marrow transplantation is not exempt of risks, and genetic engineering of autologous hHSCs is not trivial. Here, we have disrupted the CCR5 locus of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) using specific zinc finger nucleases (ZFNs) combined with homologous recombination. The modified hESCs and hiPSCs retained pluripotent characteristics and could be differentiated in vitro into CD34(+) cells that formed all types of hematopoietic colonies. Our results suggest the potential of using patient-specific hHSCs derived from ZFN-modified hiPSCs for treating AIDS.

Hairless mice have been widely applied in skin-related researches, while hairless pigs will be a useful model for skin-related study and other biomedical researches. Dickkopf-related protein 1 (DKK1) is inhibitor of Wnt signaling pathway. Transgenic mice expressing DKK1 transgene under control of a human keratin 14 (K14) promoter display hairless phenotype, which encouraged us to generate transgenic minipigs expressing pig DKK1 transgene under control of K14 promoter and finally achieve hairless minipigs. To generate transgenic cloned pigs, we constructed the lentiviral expression vector pERKDZG which contains two independent expression cassettes, the transcription of Tibet minipig DKK1 and EGFP genes are driven by K14 promoter, while mRFP is regulated under the control of Ef-1α promoter. Prior to generating the transgenic pig, the functionality of pERKDZG construct was verified by fluorescence assay and via checking pDKK1 expression. Subsequently, lentiviruses harboring ERKDZG transgene infected porcine embryonic fibroblasts (PEFs), followed by sorting RFP-positive PEFs by flow cytometry to obtain the purified PEFs carrying ERKDZG, designated DKK1-PEFs as donor cells used for somatic cell nuclear transfer (SCNT). Finally, we obtained 3 DKK1 transgenic cloned pigs with skin-specific expression of pDKK1 and EGFP transgenes, but unfortunately, DKK1 transgenic cloned pigs don't display hairless phenotype as expected. Taken together, we achieve DKK1 transgenic cloned pigs with skin-specific expression of pDKK1 transgene which provide a pig model for exploring DKK1 gene functions in pigs.

Obesity is a public health problem that increases the risk of metabolic disease, infertility, and other chronic health problems. The present study aimed to develop a new rat model for sex hormone disorder with overweight and Ca loss by intramuscular injection of exogenous leptin (LEP). Thirty female Sprague-Dawley (SD) rats (40 days old) were injected thrice intramuscularly with LEP or keyhole limpet hemocyanin immunogen. The following analyses were performed to determine the development of appetite, overweight, reproductive related-hormones, and calcium (Ca)/phosphorus (Pi) in SD rats: measurement of Lee’s index, body weight, food intake; serum Ca, Pi, and hormone tests by enzyme-linked immunosorbent analysis; histological analysis of abdominal fat; real-time polymerase chain reaction analysis of neuropeptide Y, pro-opiomelanocortin, gonadotropin-releasing hormone (Gnrh) mRNA, and gonadotropin-releasing hormone receptor (Gnrhr) mRNA expression; and western blotting analysis of enzyme phosphatidylinositol-3-kinase (PI3K). Rats injected with LEP immunogen displayed significantly increased body weight, food intake, Lee’s index, serum LEP, serum cortisol, fat deposition in the abdomen, and decreased hormones including follicle stimulating hormone, luteinizing hormone, estradiol, cholecystokinin, and Ca. Exogenous LEP administered intramuscularly also downregulate Gnrh and PI3K. In conclusion, exogenous LEP administered intramuscularly is a novel animal model for sex hormones disorder with overweight and Ca loss in SD rats. The downregulation of PI3K and Gnrh may be involved in the development of this animal model.