Generation of a genetically modified pig model with CREBRFR457Q variant.

Obesity is among the strongest risk factors for type 2 diabetes (T2D). The CREBRF missense allele rs373863828 (p. Arg457Gln, p. R457Q) is associated with increased body mass index but reduced risk of T2D in people of Pacific ancestry. To investigate the functional consequences of the CREBRF variant, we introduced the corresponding human mutation R457Q into the porcine genome. The CREBRFR457Q pigs displayed dramatically increased fat deposition, which was mainly distributed in subcutaneous adipose tissue other than visceral adipose tissue. The CREBRFR457Q variant promoted preadipocyte differentiation. The increased differentiation capacity of precursor adipocytes conferred pigs the unique histological phenotype that adipocytes had a smaller size but a greater number in subcutaneous adipose tissue (SAT) of CREBRFR457Q variant pigs. In addition, in SAT of CREBRFR457Q pigs, the contents of the peroxidative metabolites 4-hydroxy-nonenal and malondialdehyde were significantly decreased, while the activity of antioxidant enzymes, such as glutathione peroxidase, superoxide dismutase, and catalase, was increased, which was in accordance with the declined level of the reactive oxygen species (ROS) in CREBRFR457Q pigs. Together, these data supported a causal role of the CREBRFR457Q variant in the pathogenesis of obesity, partly via adipocyte hyperplasia, and further suggested that reduced oxidative stress in adipose tissue may mediate the relative metabolic protection afforded by this variant despite the related obesity.

Development of a rabbit model of Wiskott-Aldrich syndrome.

The Wiskott-Aldrich syndrome (WAS) is a severe recessive X-linked immunodeficiency resulting from loss-of-function mutations in the WAS gene. Mouse is the only mammalian model used for investigation of WAS pathogenesis. However, the mouse model does not accurately recapitulate WAS clinical phenotypes, thus, limiting its application in WAS clinical research. Herein, we report the generation of WAS knockout (KO) rabbits via embryo co-injection of Cas9 mRNA and a pair of sgRNAs targeting exons 2 and 7. WAS KO rabbits exhibited many symptoms similar to those of WAS patients, including thrombocytopenia, bleeding tendency, infections, and reduced numbers of T cell in the spleen and peripheral blood. The WAS KO rabbit model provides a new valuable tool for preclinical trials of WAS treatment.

Generation of rat blood vasculature and hematopoietic cells in rat-mouse chimeras by blastocyst complementation.

Interspecies chimera through blastocyst complementation could be an alternative approach to create human organs in animals by using human pluripotent stem cells. A mismatch of the major histocompatibility complex of vascular endothelial cells between the human and host animal will cause graft rejection in the transplanted organs. Therefore, to achieve a transplantable organ in animals without rejection, creation of vascular endothelial cells derived from humans within the organ is necessary. In this study, to explore whether donor xeno-pluripotent stem cells can compensate for blood vasculature in host animals, we generated rat-mouse chimeras by injection of rat embryonic stem cells (rESCs) into mouse blastocysts with deficiency of Flk-1 protein, which is associated with endothelial and hematopoietic cell development. We found that rESCs could differentiate into vascular endothelial and hematopoietic cells in the rat-mouse chimeras. The whole yolk sac (YS) of Flk-1EGFP/EGFP rat-mouse chimera was full of rat blood vasculature. Rat genes related to vascular endothelial cells, arteries, and veins, blood vessels formation process, as well as hematopoietic cells, were highly expressed in the YS. Our results suggested that rat vascular endothelial cells could undergo proliferation, migration, and self-assembly to form blood vasculature and that hematopoietic cells could differentiate into B cells, T cells, and myeloid cells in rat-mouse chimeras, which was able to rescue early embryonic lethality caused by Flk-1 deficiency in mouse.

Generation of Knock-In Pigs Carrying Oct4-tdTomato Reporter through CRISPR/Cas9-Mediated Genome Engineering.

The porcine pluripotent cells that can generate germline chimeras have not been developed. The Oct4 promoter-based fluorescent reporter system, which can be used to monitor pluripotency, is an important tool to generate authentic porcine pluripotent cells. In this study, we established a porcine Oct4 reporter system, wherein the endogenous Oct4 promoter directly controls red fluorescent protein (RFP). 2A-tdTomato sequence was inserted to replace the stop codon of the porcine Oct4 gene by homogenous recombination (HR). Thus, the fluorescence can accurately show the activation of endogenous Oct4. Porcine fetal fibroblast (PFF) lines with knock-in (KI) of the tdTomato gene in the downstream of endogenous Oct4 promoter were achieved using the CRISPR/CAS9 system. Transgenic PFFs were used as donor cells for somatic cell nuclear transfer (SCNT). Strong RFP expression was detected in the blastocysts and genital ridges of SCNT fetuses but not in other tissues. Two viable transgenic piglets were also produced by SCNT. Reprogramming of fibroblasts from the fetuses and piglets by another round of SCNT resulted in tdTomato reactivation in reconstructed blastocysts. Result indicated that a KI porcine reporter system to monitor the pluripotent status of cells was successfully developed.

Conversion of embryonic stem cells into extraembryonic lineages by CRISPR-mediated activators.

The recently emerged CRISPR/Cas9 technique has opened a new perspective on readily editing specific genes. When combined with transcription activators, it can precisely manipulate endogenous gene expression. Here, we enhanced the expression of endogenous Cdx2 and Gata6 genes by CRISPR-mediated activators, thus mouse embryonic stem cells (ESCs) were directly converted into two extraembryonic lineages, i.e., typical trophoblast stem cells (TSCs) and extraembryonic endoderm cells (XENCs), which exhibited characters of TSC or XENC derived from the blastocyst extraembryonic lineages such as cell morphology, specific gene expression, and differentiation ability in vitro and in vivo. This study demonstrates that the cell fate can be effectively manipulated by directly activating of specific endogenous gene expression with CRISPR-mediated activator.

Production of transgenic pigs over-expressing the antiviral gene Mx1.

The myxovirus resistance gene (Mx1) has a broad spectrum of antiviral activities. It is therefore an interesting candidate gene to improve disease resistance in farm animals. In this study, we report the use of somatic cell nuclear transfer (SCNT) to produce transgenic pigs over-expressing the Mx1 gene. These transgenic pigs express approximately 15-25 times more Mx1 mRNA than non-transgenic pigs, and the protein level of Mx1 was also markedly enhanced. We challenged fibroblast cells isolated from the ear skin of transgenic and control pigs with influenza A virus and classical swine fever virus (CFSV). Indirect immunofluorescence assay (IFA) revealed a profound decrease of influenza A proliferation in Mx1 transgenic cells. Growth kinetics showed an approximately 10-fold reduction of viral copies in the transgenic cells compared to non-transgenic controls. Additionally, we found that the Mx1 transgenic cells were more resistant to CSFV infection in comparison to non-transgenic cells. These results demonstrate that the Mx1 transgene can protect against viral infection in cells of transgenic pigs and indicate that the Mx1 transgene can be harnessed to develop disease-resistant pigs.

Generation of multi-gene knockout rabbits using the Cas9/gRNA system.

The prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) is a simple, robust and efficient technique for gene targeting in model organisms such as zebrafish, mice and rats. In this report, we applied CRISPR technology to rabbits by microinjection of Cas9 mRNA and guided RNA (gRNA) into the cytoplasm of pronuclear-stage embryos. We achieved biallelic gene knockout (KO) rabbits by injection of 1 gene (IL2rg) or 2 gene (IL2rg and RAG1) Cas9 mRNA and gRNA with an efficiency of 100%. We also tested the efficiency of multiple gene KOs in early rabbit embryos and found that the efficiency of simultaneous gene mutation on target sites is as high as 100% for 3 genes (IL2rg, RAG1 and RAG2) and 33.3% for 5 genes (IL2rg, RAG1, RAG2, TIKI1 and ALB). Our results demonstrate that the Cas9/gRNA system is a highly efficient and fast tool not only for single-gene editing but also for multi-gene editing in rabbits.

Establishment of a rabbit Oct4 promoter-based EGFP reporter system.

Rabbits are commonly used as laboratory animal models to investigate human diseases and phylogenetic development. However, pluripotent stem cells that contribute to germline transmission have yet to be established in rabbits. The transcription factor Oct4, also known as Pou5f1, is considered essential for the maintenance of the pluripotency of stem cells. Hence, pluripotent cells can be identified by monitoring Oct4 expression using a well-established Oct4 promoter-based reporter system. This study developed a rabbit Oct4 promoter-based enhanced green fluorescent protein (EGFP) reporter system by transfecting pROP2-EGFP into rabbit fetal fibroblasts (RFFs). The transgenic RFFs were used as donor cells for somatic cell nuclear transfer (SCNT). The EGFP expression was detected in the blastocysts and genital ridges of SCNT fetuses. Fibroblasts and neural stem cells (NSCs) were derived from the SCNT fetuses. EGFP was also reactivated in blastocysts after the second SCNT, and induced pluripotent stem cells (iPSCs) were obtained after reprogramming using Yamanaka's factors. The results above indicated that a rabbit reporter system used to monitor the differentiating status of cells was successfully developed.

RAG1/2 knockout pigs with severe combined immunodeficiency.

Pigs share many physiological, biochemical, and anatomical similarities with humans and have emerged as valuable large animal models for biomedical research. Considering the advantages in immune system resemblance, suitable size, and longevity for clinical practical and monitoring purpose, SCID pigs bearing dysfunctional RAG could serve as important experimental tools for regenerative medicine, allograft and xenograft transplantation, and reconstitution experiments related to the immune system. In this study, we report the generation and phenotypic characterization of RAG1 and RAG2 knockout pigs using transcription activator-like effector nucleases. Porcine fetal fibroblasts were genetically engineered using transcription activator-like effector nucleases and then used to provide donor nuclei for somatic cell nuclear transfer. We obtained 27 live cloned piglets; among these piglets, 9 were targeted with biallelic mutations in RAG1, 3 were targeted with biallelic mutations in RAG2, and 10 were targeted with a monoallelic mutation in RAG2. Piglets with biallelic mutations in either RAG1 or RAG2 exhibited hypoplasia of immune organs, failed to perform V(D)J rearrangement, and lost mature B and T cells. These immunodeficient RAG1/2 knockout pigs are promising tools for biomedical and translational research.

Direct conversion of human fibroblasts into neuronal restricted progenitors.

Neuronal restricted progenitors (NRPs) represent a type of transitional intermediate cells that lie between multipotent neural progenitors and terminal differentiated neurons during neurogenesis. These NRPs have the ability to self-renew and differentiate into neurons, but not into glial cells, which is considered an advantage for cellular therapy of human neurodegenerative diseases. However, difficulty in the extraction of highly purified NRPs from normal nervous tissue prevents further studies and applications. In this study, we report the conversion of human fetal fibroblasts into human induced NRPs (hiNRPs) in 11 days by using just three defined factors: Sox2, c-Myc, and either Brn2 or Brn4. The hiNRPs exhibited distinct neuronal characteristics, including cell morphology, multiple neuronal marker expression, self-renewal capacity, and a genome-wide transcriptional profile. Moreover, hiNRPs were able to differentiate into various terminal neurons with functional membrane properties but not glial cells. Direct generation of hiNRPs from somatic cells will provide a new source of cells for cellular replacement therapy of human neurodegenerative diseases.

Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs.

Inbred mini-pigs are ideal organ donors for future human xenotransplantations because of their clear genetic background, high homozygosity, and high inbreeding endurance. In this study, we chose fibroblast cells from a highly inbred pig line called Banna mini-pig inbred line (BMI) as donor nuclei for nuclear transfer, combining with transcription activator-like effector nucleases (TALENs) and successfully generated α-1,3-galactosyltransferase (GGTA1) gene biallelic knockout (KO) pigs. To validate the efficiency of TALEN vectors, in vitro-transcribed TALEN mRNAs were microinjected into one-cell stage parthenogenetically activated porcine embryos. The efficiency of indel mutations at the GGTA1-targeting loci was as high as 73.1% (19/26) among the parthenogenetic blastocysts. TALENs were co-transfected into porcine fetal fibroblasts of BMI with a plasmid containing neomycin gene. The targeting efficiency reached 89.5% (187/209) among the survived cell clones after a 10 d selection. More remarkably 27.8% (58/209) of colonies were biallelic KO. Five fibroblast cell lines with biallelic KO were chosen as nuclear donors for somatic cell nuclear transfer (SCNT). Three miniature piglets with biallelic mutations of the GGTA1 gene were achieved. Gal epitopes on the surface of cells from all the three biallelic KO piglets were completely absent. The fibroblasts from the GGTA1 null piglets were more resistant to lysis by pooled complement-preserved normal human serum than those from wild-type pigs. These results indicate that a combination of TALENs technology with SCNT can generate biallelic KO pigs directly with high efficiency. The GGTA1 null piglets with inbred features created in this study can provide a new organ source for xenotransplantation research.

Expression of plant sweet protein brazzein in the milk of transgenic mice.

Sugar, the most popular sweetener, is essential in daily food. However, excessive sugar intake has been associated with several lifestyle-related diseases. Finding healthier and more economical alternatives to sugars and artificial sweeteners has received increasing attention to fulfill the growing demand. Brazzein, which comes from the pulp of the edible fruit of the African plant Pentadiplandra brazzeana Baill, is a protein that is 2,000 times sweeter than sucrose by weight. Here we report the production of transgenic mice that carry the optimized brazzein gene driven by the goat Beta-casein promoter, which specifically directs gene expression in the mammary glands. Using western blot analysis and immunohistochemistry, we confirmed that brazzein could be efficiently expressed in mammalian milk, while retaining its sweetness. This study presents the possibility of producing plant protein-sweetened milk from large animals such as cattle and goats.

Ankylosis of temporomandibular joint after the traumatic brain injury: a report of two cases.

Mouth opening limitation after the neurosurgical procedures is a common complication and usually resolves within 3 months. If limited mouth opening remains unresolved on the long term, an intra-articular ankylosis of temporomandibular joint may develop eventually. The possible mechanisms base on the myositis and atrophy of the masticatory muscles for these craniotomies are often involved in the temporalis. This article reports two unusual cases with the intra-articular ankylosis of temporomandibular joint after the traumatic brain injury, who received a modified surgical treatment for joint ankylosis. Therefore, the early diagnosis and intervention are important to minimize these complications.

Immediate autotransplantation of mandibular third molar in China.

OBJECTIVE: Tooth autotransplantation is a useful surgical method to replace a nonrestorable tooth. We reported our experiences in the replacement of mandibular nonrestorable molars by immediate autotransplantation in a Chinese population. STUDY DESIGN: Thirty-five mandibular third molars with open or closed apices from 34 patients were autotransplanted into the same or contralateral fresh recipient sites immediately after the extraction of the nonrestorable mandibular molars. Root canal treatment was routinely performed in the closed-apical molars within 1 month after surgery. Clinical and radiographic examination of the transplanted donor molars was done after surgery. RESULTS: Two teeth were been extracted for progressive root resorption. The remaining 33 autotransplanted teeth were asymptomatic and functioning after a mean follow-up period of 5.2 years. No infection, ankylosis, loss of the transplants, or root resorption was noted in the remaining autotransplanted teeth. CONCLUSION: Immediate autotransplantation of the mandibular third molar is a reasonable and alternative treatment to replace a nonrestorable tooth in China.

Vimentin affects the mobility and invasiveness of prostate cancer cells.

A significant proportion of prostate cancer patients treated with curative intent go on to develop advanced disease. At a fundamental biological level, very little is known about what makes the disease aggressive and metastatic. Observational pathology reports and experimental data suggest that epithelial-mesenchymal transition is involved in prostate cancer invasiveness. Here, we investigated vimentin expression of prostate cancer cells, and explored the potential mechanism of vimentin promoting prostate cancer cells invasion. Vimentin expression was not detected in well differentiated tumors or in moderately differentiated tumors, but the majority of poorly differentiated cancers (5/11 with negative bone scan, 11/14 bone with positive scan) and bone metastases (8/8) had high vimentin expression in tumor cells. Downregulation of vimentin expression in PC-3 cells by transfection with antisense-vimentin led to a significant decrease in tumor cells motility and invasive activity. Furthermore, the expression of E-cadherin was inversely associated with expression of vimentin. Our results suggest that vimentin affects prostate cancer cells motility and invasiveness.