Yifan Dai

Generation of B Cell-Deficient Pigs by Highly Efficient CRISPR/Cas9-Mediated Gene Targeting

Generating B cell-deficient mutant is the first step to produce human antibody repertoires in large animal models. In this study, we applied the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system to target the JH region of the pig IgM heavy chain gene which is crucial for B cell development and differentiation. Transfection of IgM-targeting Cas9 plasmid in primary porcine fetal fibroblasts (PFFs) enabled inducing gene knock out (KO) in up to 53.3% of colonies analyzed, a quarter of which harbored biallelic modification, which was much higher than that of the traditional homologous recombination (HR). With the aid of somatic cell nuclear transfer (SCNT) technology, three piglets with the biallelic IgM heavy chain gene mutation were produced. The piglets showed no antibody-producing B cells which indicated that the biallelic mutation of the IgM heavy chain gene effectively knocked out the function of the IgM and resulted in a B cell-deficient phenotype. Our study suggests that the CRISPR/Cas9 system combined with SCNT technology is an efficient genome-editing approach in pigs.

Derivation of Porcine Embryonic Stem-Like Cells from In Vitro-Produced Blastocyst-Stage Embryos

Efficient isolation of embryonic stem (ES) cells from pre-implantation porcine embryos has remained a challenge. Here, we describe the derivation of porcine embryonic stem-like cells (pESLCs) by seeding the isolated inner cell mass (ICM) from in vitro-produced porcine blastocyst into α-MEM with basic fibroblast growth factor (bFGF). The pESL cells kept the normal karyotype and displayed flatten clones, similar in phenotype to human embryonic stem cells (hES cells) and rodent epiblast stem cells. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers such as OCT4, NANOG, SOX2, SSEA-4, TRA-1-60, and TRA-1-81 as determined by both immunofluorescence and RT-PCR. Additionally, these cells formed embryoid body (EB), teratomas and also differentiated into 3 germ layers in vitro and in vivo. Microarray analysis showed the expression of the pluripotency markers, PODXL, REX1, SOX2, KLF5 and NR6A1, was significantly higher compared with porcine embryonic fibroblasts (PEF), but expression of OCT4, TBX3, REX1, LIN28A and DPPA5, was lower compared to the whole blastocysts or ICM of blastocyst. Our results showed that porcine embryonic stem-like cells can be established from in vitro-produced blastocyst-stage embryos, which promote porcine naive ES cells to be established.

Generation of complement protein C3 deficient pigs by CRISPR/Cas9-mediated gene targeting

Complement protein C3 is the pivotal component of the complement system. Previous studies have demonstrated that C3 has implications in various human diseases and exerts profound functions under certain conditions. However, the delineation of pathological and physiological roles of C3 has been hampered by the insufficiency of suitable animal models. In the present study, we applied the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system to target the C3 gene in porcine fetal fibroblasts. Our results indicated that CRISPR/Cas9 targeting efficiency was as high as 84.7%, and the biallelic mutation efficiency reached at 45.7%. The biallelic modified colonies were used as donor for somatic cell nuclear transfer (SCNT) technology to generate C3 targeted piglets. A total of 19 C3 knockout (KO) piglets were produced and their plasma C3 protein was undetectable by western blot analysis and ELISA. The hemolytic complement activity and complement-dependent cytotoxicity assay further confirmed that C3 was disrupted in these piglets. These C3 KO pigs could be utilized as a valuable large animal model for the elucidation of the roles of C3.

Overexpressing dominant-negative FGFR2-IIIb impedes lung branching morphogenesis in pigs

Genetic studies with mouse models have shown that fibroblast growth factor receptor 2-IIIb (FGFR2-IIIb) plays crucial roles in lung development and differentiation. To evaluate the effect of FGFR2-IIIb in pig lung development, we employed somatic cell nuclear transfer (SCNT) technology to generate transgenic pig fetuses overexpressing the transmembrane (dnFGFR2-IIIb-Tm) and soluble (dnFGFR2-IIIb-HFc) forms of the dominant-negative human FGFR2-IIIb driven by the human surfactant protein C (SP-C) promoter, which was specifically expressed in lung epithelia. Eight dnFGFR2-IIIb-Tm transgenic and twelve dnFGFR2-IIIb-HFc transgenic pig fetuses were collected from three and two recipient sows, respectively. Repression of FGFR2-IIIb in lung epithelia resulted in smaller lobes and retardation of alveolarization in both forms of dnFGFR2-IIIb transgenic fetuses. Moreover, the dnFGFR2-IIIb-HFc transgenic ones showed more deterioration in lung development. Our results demonstrate that disruption of FGFR2-IIIb signaling in the epithelium impedes normal branching and alveolarization in pig lungs, which is less severe than the results observed in transgenic mice. The dnFGFR2-IIIb transgenic pig is a good model for the studies of blastocyst complementation as well as the mechanisms of lung development and organogenesis.

Corrigendum: Omega-3 Polyunsaturated Fatty Acids Attenuate Fibroblast Activation and Kidney Fibrosis Involving MTORC2 Signaling Suppression

This corrects the article DOI: 10.1038/srep46146.

Genome-wide screen for serum microRNA expression profile in mfat-1 transgenic mice

Abstractn-3 Polyunsaturated fatty acids (n-3 PUFAs) contribute to preventing many types of diseases, including cancer; however, a high n-6 polyunsaturated fatty acids (n-6 PUFAs) intake in modern diets has the opposite effect. Previously, we developed a transgenic mouse model that expresses a gene, fat-1, encoding an n-3 fatty acid desaturase, which converts n-6 PUFAs to n-3 PUFAs in vivo. MicroRNAs (miRNAs) in serum are stable, reproducible, and consistent among individuals of the same species and serve as potential biomarkers for the detection of cancers and other diseases. Employing illumina sequencing, we analyzed all the serum miRNAs in wild-type and mfat-1 transgenic mice. Using quantitative real-time PCR (RT-qPCR), we identified 12 miRNAs that were highly expressed in mfat-1 mice. Pathway analysis of targets regulated by these miRNAs revealed a significant number of genes involved in the development of cancer, including phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinases (MAPK), and mammalian target of rapamycin (mTOR), which suggested a relationship between n-3 PUFAs and cancer prevention.

mfat-1 transgene protects cultured adult neural stem cells against cobalt chloride–mediated hypoxic injury by activating Nrf2/ARE pathways

Ischemic stroke is a devastating neurological disorder and one of the leading causes of death and serious disability in adults. Adult neural stem cell (NSC) replacement therapy is a promising treatment for both structural and functional neurological recovery. However, for the treatment to work, adult NSCs must be protected against hypoxic‐ischemic damage in the ischemic penumbra. In the present study, we aimed to investigate the neuroprotective effects of the mfat‐1 transgene on cobalt chloride (CoCl2)‐induced hypoxic‐ischemic injury in cultured adult NSCs as well as its underlying mechanisms. The results show that in the CoCl2‐induced hypoxic‐ischemic injury model, the mfat‐1 transgene enhanced the viability of adult NSCs and suppressed CoCl2‐mediated apoptosis of adult NSCs. Additionally, the mfat‐1 transgene promoted the proliferation of NSCs as shown by increased bromodeoxyuridine labeling of adult NSCs. This process was related to the reduction of reactive oxygen species. Quantitative real‐time polymerase chain reaction and Western blot analysis revealed a much higher expression of nuclear factor erythroid 2‐related factor 2 (Nrf2) and its downstream genes (HO‐1, NQO‐1, GCLC). Taken together, our findings show that the mfat‐1 transgene restored the CoCl2‐inhibited viability and proliferation of NSCs by activating nuclear factor erythroid 2‐related factor 2 (Nrf2)/antioxidant response elements (ARE) signal pathway to inhibit oxidative stress injury. Further investigation of the function of the mfat‐1 transgene in adult protective mechanisms may accelerate the development of adult NSC replacement therapy for ischemic stroke.

Apolipoprotein E deficiency accelerates atherosclerosis development in miniature pigs

ABSTRACT Miniature pigs have advantages over rodents in modeling atherosclerosis because their cardiovascular system and physiology are similar to that of humans. Apolipoprotein E (ApoE) deficiency has long been implicated in cardiovascular disease in humans. To establish an improved large animal model of familial hypercholesterolemia and atherosclerosis, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 system (CRISPR/Cas9) was used to disrupt the ApoE gene in Bama miniature pigs. Biallelic-modified ApoE pigs with in-frame mutations (ApoEm/m) and frameshift mutations (ApoE−/−) were simultaneously produced. ApoE−/− pigs exhibited moderately increased plasma cholesterol levels when fed with a regular chow diet, but displayed severe hypercholesterolemia and spontaneously developed human-like atherosclerotic lesions in the aorta and coronary arteries after feeding on a high-fat and high-cholesterol (HFHC) diet for 6u2005months. Thus, these ApoE−/− pigs could be valuable large animal models for providing further insight into translational studies of atherosclerosis. Editors choice: ApoE knockout pigs displayed severe hypercholesterolemia and spontaneously developed human-like atherosclerotic lesions in the aorta and coronary arteries within 6 months of feeding on a high-fat and high-cholesterol diet.

DNA demethylation pattern of in-vitro fertilized and cloned porcine pronuclear stage embryos

Recent studies in mice showed that the Ten-eleven translocation Enzymes (TET) family is involved in the active DNA demethylation. The isotype TET-3 is responsible for the conversion of 5mc (5-methylcytosine) to 5hmc (5-hydroxymethylcytosine) at the pronuclear stages of mouse embryo. This study was performed to investigate the pattern of methylation change and the role of TET family in the demethylation process of porcine in-vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) derived embryo. Bisulfite-sequencing PCR (BSP) and DNA glucosylation and digestion before quantitative PCR (qGluMS-PCR) were done to evaluate the exact change of methylation during porcine pronuclear stages. The results showed that the amount of 5hmc detected increased whereas 5mc decreased in IVF embryo from pronuclear stage 2 (PN2) to pronuclear stage 5 (PN5). In addition, Immunofluorescent staining showed that the 5hmc signal, also detected in oocytes, significantly increased in both pronucleus from fertilization to PN2. The amount of 5hmc continued to rise in male pronucleus but decreased to a very low level in female pronucleus from PN2 to PN5. The above results indicate that female pronucleus might undergo active demethylation only at early pronuclear stages. On the other hand, male pronucleus might undergo active demethylation throughout all pronuclear stages. The expression of three TET isotypes (TET-1, TET-2, TET-3) were tested and TET-3 was found to be the highest expressed isotype. High TET-3 concentrations observed mainly in male pronucleus using immunofluorescent staining, implying that TET-3 might be the main enzyme which catalyzes the conversion of 5mc to 5hmc. In contrast, no TET-3 signal was detected in female pronucleus through the pronuclear stages. The demethylation pattern of SCNT embryos resembled that of the male pronucleus of IVF embryos, suggesting that active demethylation might happen in porcine cloned embryo.

Generation of monoclonal antibodies against n-3 fatty acid desaturase.

Dear Editor: n nOmega-3 polyunsaturated fatty acids (n-3 PUFAs) are essential fatty acids for normal cellular functions and have been used for prevention and treatment of many diseases, including coronary heart disease, diabetes, and cancers[1-3]. n-3 PUFAs and n-6 PUFAs have been shown to decrease and increase the severity of several human diseases, respectively[4]. Unfortunately, mammals have no enzymes to synthesize n-6 and n-3 fatty acids. Therefore, they must rely on a dietary supply. Today, the ratio of n-6 to n-3 PUFAs has reached to an unhealthy 20-30:1 due to people taking n-6 rich diet and eating less sea fish. Caenorhabditis (C.) elegans fat-1 gene encodes an n-3 desaturase that introduces a double bond at n-3 position of n-6 fatty acids to form n-3 fatty acids[5]. Several fat-1 transgenic mammals (including mouse, pig and cow) have been produced in which n-3 PUFAs were increased and n-6 PUFAs were reduced, resulting in a significant reduction of n-6/n-3 ratio in those transgenic animals[6-8]. However, due to lack of commercially available FAT-1 antibody, FAT-1 expression is indirectly determined by measurement of FAT-1 activity including increase of n-3 PUFAs and decrease of n-6/n-3 ratio in animals. In this study, we used recombinant FAT-1 to immunize mice and to generate FAT-1 specific monoclonal antibody. Then, FAT-1 monoclonal antibodies were used to detect FAT-1 protein in tissue samples of the liver, lung, kidney, heart, brain, and spleen of fat-1 transgenic mice by Western blotting and immunohistochemical staining assays. n nEscherichia (E.) coli expression system allows rapid and economical production of recombinant FAT-1 protein. To express C. elegans fat-1 gene in E. coli efficiently, we optimized the codons of the full-length C. elegans fat-1 gene sequence using GeneArt® Gene Synthesis (GeneArt, Regensburg, Germany) followed by subcloning into the expression vector pCold II. The recombinant FAT-1 protein was purified by HisTrap FF affinity chromatography column (GE Healthcare Life Sciences, Piscataway, NJ, USA) and analyzed by SDS-PAGE and Coomassie brilliant blue staining (Fig. 1A), which showed a protein band of approximately 46 kDa in size, corresponding to the known molecular weight of FAT-1[5]. The identity of the FAT-1 recombinant protein was further confirmed by searching sequence databases using mass spectrometry data. Recombinant FAT-1 was used to generate FAT-1 monoclonal antibodies and after three rounds of subcloning, the subclone 3A11 showed the best titer. Coomassie brilliant blue staining revealed the presence of two protein bands 55 kDa and 25 kDa in size, respectively (Fig. 1B). Enzyme-linked immunosorbent assay (ELISA) further showed that the FAT-1 monoclonal antibodies (3A11) between 0.05 and 100 μg/mL exhibited a linear increase in OD450 (Fig. 1C). Further analysis revealed that the antibodies were IgG2a. n n n nFig. 1 n nPreparation of FAT-1 monoclonal antibodies. n n n nImmunoblotting assays using FAT-1 monoclonal antibodies (3A11) demonstrated a protein band of approximately 46 kDa in size from the homogenates of tissue specimens of the liver, lung, kidney, heart, brain, and spleen of mfat-1 transgenic mice, but not from those of normal C57BL/6 mice (Fig. 2A). A protein band approximately 26 kDa in size was also detected, which may be FAT-1 degradation products. FAT-1 is rich in cysteine and has three successive cysteines at positions 241-243. As the two disulfide bonds can be broken under denaturing conditions, the stability of peptide bond may be affected subsequently. Immunohistochemical staining of tissue specimens of transgenic and normal C57BL/6 mice further demonstrated that FAT-1 protein was present in many tissues of the transgenic mice, but was not found in the wildtype control C57BL/6 tissues (Fig. 2B-O). n n n nFig. 2 n nFunction of FAT-1 monoclonal antibodies. n n n nThe expression of fat-1 gene or FAT-1 in transgenic tissues or cells can be examined previously only by reverse transcription-polymerase chain reaction or by gas chromatography to measure the increase of n-3 PUFAs and the decrease of n-6/n-3 ratios. The generation of FAT-1 monoclonal antibodies provides a new tool to detect FAT-1 protein directly. n nThis work was supported by grants from the National Natural Science Foundation of China (81202370) and Jiangsu Key Laboratory of Xenotransplantation (BM2012116). Yifan Dai is a Fellow at the Collaborative Innovation Center For Cardiovascular Disease Translational Medicine. n nYours Sincerely,