Publications (16)

  • [Show abstract] [Hide abstract] ABSTRACT: Cytoplasmic microinjection (CI) of foreign gene into in vivo fertilized zygotes has emerged as a useful tool for transgenic pig production. In the current study, we investigated factors affecting transgenic efficiency and developmental potential of parthenogenetic (PA) and in vitro-fertilized (IVF) porcine embryos produced by CI. These factors included adding of RNase inhibitor, DNA or RNA concentration, injection time, and different structures of plasmids. Our results showed that adding of 1-4 U/μL of RNase inhibitor did not have negative effect on development potential of CI-PA embryos, and RNase inhibitor injection significantly increased EGFP expressing rate of CI-PA embryos. High injection DNA concentration and long injection interval after PA significantly reduced blastocyst formation. Different molecular structures such as DNA or RNA affected CI-PA embryos development, and RNA had little harmful effect on pig's early embryonic development. EGFP expression rate of CI-IVF embryos was improved following the increase of foreign DNA concentration, but blastocyst formation rate was decreased. Injection time after IVF did not show any significant difference on embryonic development, but longer interval resulted in a significantly lower EGFP expressing rate. Cas9 mRNA and myostatin (GDF-8) sgRNA co-injection indicated that the mutation rate of CI-IVF group was significantly higher than that of CI-PA. The CI-IVF-generated embryos were then transferred to six recipient pigs, but no live piglets were obtained. The following pronuclear formation assessment showed more than 76.1% IVF zygotes were polyspermy. These results demonstrate that CI-PA and CI-IVF were effective methods for production of transgenic pig embryos. However, polyspermic fertilization and poor quality of porcine IVF blastocysts are still the main problem of resulting in pregnancy failure.
    Article · Apr 2016 · In Vitro Cellular & Developmental Biology - Animal
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    Wanyou Feng · Shibei Shibei Chen · Qingyou Liu · [...] · Deshun Shi
    [Show abstract] [Hide abstract] ABSTRACT: Isolation and culture of spermatogonial stem cells (SSCs) are attractive for production of genetic modified offspring. In the present study, buffalo SSCs were isolated, cultured and expression pattern of different germ cell marker genes were determined. To recover spermatogonia, testes from age 3 to 7 months of buffalo were decapsulated, and seminiferous tubules were enzymatically dissociated. Two types of cells, immature sertoli cell and type A spermatogonia were observed in buffalo testes in this stage. Germ cell marker genes, OCT3/4 (Pou5f1), THY-1, c-kit, PGP9.5 (UCHL-1) and DBA, were determined to be expressed both in mRNA and protein level by RT-PCR and immunostaining in buffalo testes and SSCs, respectively. In the following, when the isolated buffalo SSCs were cultured in the medium supplemented 2.5% FBS and 40ng/ml GDNF medium, SSCs proliferation efficiency and colony number were significantly improved than those of other groups (p<0.05). These findings may help in isolation and establishing long term in vitro culture system for buffalo SSCs, and accelerating the generation of genetic modified buffaloes.
    Full-text available · Article · Nov 2015 · Asian Australasian Journal of Animal Sciences
  • [Show abstract] [Hide abstract] ABSTRACT: BMP-1/TLD-related metalloproteinases play a key role in morphogenesis via the proteolytic maturation of a number of ECM proteins and the activation of a subset of growth factors of the TGF-β superfamily. Recent data indicated that BMP-1 is expressed in sheep ovarian follicles and showed a protease activity. The aim of the current study was to characterize the function of the buffalo BMP-1 gene in folliculogenesis. A 3195 bp buffalo BMP-1 mRNA fragment was firstly cloned and sequenced, which contained a whole 2967 bp CDS. The multialigned results suggested that BMP-1 is highly conserved among different species both at the nucleic acid and the amino acid level. BMP-1 is located in the oogonium of the fetal buffalo ovary, as well as in the granulosa cells (GCs) and the oocytes of adult ovary from the primordial to the large antral follicles. Further study showed that BMP-1 promoted cell cycle and proliferation and inhibited apoptosis in in vitro cultured GCs. Adding BMP-1 recombinant protein to the culture medium of the GCs increased the expression of the key cell cycle regulators such as Cyclin D1 and Cyclin D2. And down-regulated the expression of cell apoptosis pathway genes such as Cytochrome C, Fas, FasL and Chop .etc, both at the mRNA and the protein levels. It also up-regulated the expression of PAPP-A, IGF system and VEGF etc., which play important roles in the selection and dominance of growth follicles. The opposite results were observed by adding BMP-1 antibody to the investigation groups. This study suggests that BMP-1 regulates the proliferation and apoptosis of in vitro cultured GCs by changing the expression pattern of related genes, and may potentially promote the selection and dominance of the buffalo follicles.
    Article · Nov 2015 · Theriogenology
  • [Show abstract] [Hide abstract] ABSTRACT: Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven‑hoofed animals, which leads to serious economical losses. FMDV is not adequately controlled by vaccination or biosecurity measures. To generate genetically modified FMDV‑resistant animals, a combinatorial expression cassette producing three short hairpin (sh)RNAs was constructed using the lentivirus (LV) vector, LV‑3shRNA. The three shRNAs were expressed under the regulation of DNA polymerase III promoters from a buffalo and a bovine source, with one targeted to the non‑structural protein 3B, and the other two targeted to the viral polymerase protein 3D of FMDV, respectively. The role of LV‑3shRNA in the inhibition of the replication of FMDV was determined in BHK‑21 cells and in suckling mice. The results revealed that LV‑3shRNA reduced viral growth 3‑fold (24 h post‑infection) when the cells were challenged with 107‑times the tissue culture infective dose (TCID50)/ml of O serotype FMDV. The suckling mice pretreated with LV‑3shRNA were completely protected on administration of 5‑times the dose of FMDV otherwise sufficient to kill 50% of the experimental animals (LD50). These results demonstrated that the LV‑mediated dual expression of three FMDV‑specific shRNAs provided a novel strategy towards combating FMDV, which facilitates the permanent introduction of novel disease-resistance traits into the buffalo and bovine genomes in the future.
    Article · Aug 2015 · Molecular Medicine Reports
  • [Show abstract] [Hide abstract] ABSTRACT: The present study was undertaken to examine the effect of Scriptaid treatment on histone acetylation, DNA methylation, expression of genes related to histone acetylation, and development of buffalo somatic cell nuclear transfer (SCNT) embryos. Treatment of buffalo SCNT embryos with 500 nM Scriptaid for 24 h resulted in a significant increase in the blastocyst formation rate (28.2% vs. 13.6%, p<0.05). Meanwhile, treatment of buffalo SCNT embryos with Scriptaid also resulted in higher acetylation levels of H3K18 and lower methylation levels of global DNA at the blastocyst stage, which was similar to fertilized counterparts. The expression levels of CBP, p300, HAT1, Dnmt1, and Dnmt3a in SCNT embryos treated with Scriptaid were significantly lower than the control group at the eight-cell stage (p<0.05), but the expression of HAT1 and Dnmt1a was higher than the control group at the blastocyst stage (p<0.05). When 96 blastocysts developed from Scriptaid-treated SCNT embryos were transferred into 48 recipients, 11 recipients (22.9%) became pregnant, whereas only one recipient (11.1%) became pregnant following transfer of 18 blastocysts developed from untreated SCNT embryos into nine recipients. These results indicate that treatment of buffalo SCNT embryos with Scriptaid can improve their developmental competence, and this action is mediated by resulting in a similar histone acetylation level and global DNA methylation level compared to in vitro-fertilized embryos through regulating the expression pattern of genes related to histone acetylation and DNA methylation.
    Article · Jun 2015
  • Yanping Ren · Xiangping Li · Qingyou Liu · [...] · Deshun Shi
    [Show abstract] [Hide abstract] ABSTRACT: Reporter genes are often used as markers to track the integration and expression of target genes in animal genetic engineering. To avoid potential side effects from reporter genes, in this study an improved Tet-on system was developed to control reporter gene expression, and its effectiveness was explored in transgenic cells. First, the rtTA protein was fused with Tat and NLS proteins to obtain the prokaryotic expression vector pET32a-Tat-rtTA-NSL. A eukaryotic transgenic vector was constructed, p-HS4-BPA-TmA-HS4, in which the reporter (mCherry) and target (PRL) genes were promoted by TRE and BCN, respectively. After confirming the functionality of the transgenic vector, purified rtTA protein and Dox were added to induce expression of the mCherry gene. The optimal amount of purified rtTA protein, its influence on target gene expression, and the time of rtTA protein action were each investigated separately. The results showed that rtTA protein was expressed in transformed E. coli with IPTG induction. TRE could promote mCherry gene expression by co-transfecting the constructed transgenic vector and prtTA plasmid. When purified rtTA protein and Dox were added, red fluorescence was observed in Bcap-37 cells transfected with the p-HS4-BPA-TmA-HS4 vector, and the exogenous PRL gene was expressed regardless of mCherry gene expression. The optimal amount of rtTA protein was 16 μg/mL, and it needed about 6 h to promote mCherry gene expression in transfected cells. These results demonstrate that the expression of the mCherry reporter gene can be tightly and conditionally regulated by our Tet-on system.
    Article · Feb 2015 · Biotechnology and Bioprocess Engineering
  • Article · Jan 2015 · Stem Cell Discovery
  • Shun Zhang · Fenghua Lu · Qingyou Liu · [...] · Deshun Shi
    [Show abstract] [Hide abstract] ABSTRACT: N-3 polyunsaturated fatty acids (n-3 PUFAs) have their first double bond at the third carbon from the methyl end of the fatty-acid chain and had been proven to be beneficial to human health. However, mammals cannot produce n-3 PUFAs by themselves because they lack the n-3 fatty-acid desaturase (Fat-1) gene. Thus, the possibility of producing sFat-1 transgenic rabbits was explored in this study. The transgenic cassette of pPGK1-sFat-1-CMV-EGFP was constructed and transgenic rabbit embryos were produced by intracytoplasmic sperm injection (ICSI). When 123 EGFP-positive embryos at the 2-8-cell stage were transplanted into the oviduct of four oestrous-synchronised recipients, two of them became pregnant and gave birth to seven pups. However, transfer of embryos into the uterus of oestrous-synchronised recipients and oviduct or uterus of oocyte donor rabbits did not result in pregnancy. The integration of the sFat-1 gene was confirmed in six of the seven live pups by PCR and Southern blot. The expression of the sFat-1 gene in the six transgenic pups was also detected by reverse transcription polymerase chain reaction (RT-PCR). Gas chromatography-mass spectrometry analysis revealed that transgenic rabbits exhibited an ~15-fold decrease in the ratio of n-6:n-3 PUFAs in muscle compared with wild-type rabbits and non-transgenic rabbits. These results demonstrate that sFat-1 transgenic rabbits can be produced by ICSI and display a low ratio of n-6:n-3 PUFAs.
    Article · Jul 2014 · Reproduction Fertility and Development
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    Xiaoxi Zhang · Qingyou Liu · Chan Luo · [...] · Deshun Shi
    [Show abstract] [Hide abstract] ABSTRACT: RNA polymerase III (pol III) type 3 promoters, such as 7SK and U6, are routinely used to induce short hairpin RNAs (shRNAs) to knockdown gene expression by RNA interference (RNAi). To extend the application of RNAi to studies of buffalo, an shRNAs expressing system using the buffalo pol III promoters was developed. Buffalo 7SK promoter (bu7SK) and U6 promoter (buU6) sequences upstream of the full-length 7SK and U6 small nuclear RNA sequence in the buffalo genome were identified and characterized, respectively. To determine the functionality of these promoters in constructs driving shRNA expression, anti-EGFP shRNAs (shEGFP) cassettes under the direction of bu7SK and buU6 were constructed. We further compared the EGFP knockdown efficiency of constructs using bu7SK and buU6 with that of promoters of human and bovine origins in BFF cells and mouse PT67 cells by flow cytometry and quantitative real-time PCR assays. We found that the bu7SK and buU6 promoters induced the greatest level of suppression in homologous and heterologous cells relative to promoters derived from other species. Taken together, functional bu7SK and buU6 promoters were identified and characterized, thus laying the groundwork for future development of RNAi therapeutics and gene modification in buffalo species.
    Full-text available · Article · Feb 2014 · International Journal of Molecular Sciences
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    [Show abstract] [Hide abstract] ABSTRACT: It has been reported that buffalo (Bubalus bubalis) embryos reconstructed by somatic cell nucleus transfer (SCNT) can develop to the full term of gestation and result in newborn calves. However, the developmental competence of reconstructed embryos is still low. Recently, it has been reported that treating donor cells or embryos with trichostatin A (TSA) can increase the cloning efficiency in some species. Thus, the present study was undertaken to improve the development of buffalo SCNT embryos by treatment of donor cells (buffalo fetal fibroblasts) with TSA and explore the relation between histone acetylation status of donor cells and developmental competence of SCNT embryos. Treatment of donor cells with either 0.15 or 0.3 μM TSA for 48 hours resulted in a significant increase in the cleavage rate and blastocyst yield of SCNT embryos (P < 0.05). Meanwhile, the expression level of HDAC1 in donor cells was also decreased (0.4-0.6 fold, P < 0.05) by TSA treatment, although the expression level of HAT1 was not affected. Further measurement of the epigenetic maker AcH4K8 in buffalo IVF and SCNT embryos at the eight-cell stage revealed that the spatial distribution of acH4K8 staining in SCNT embryos was different from the IVF embryos. Treatment of donor cells with TSA resulted in an increase in the AcH4K8 level of SCNT embryos and similar to fertilized counterparts. These results suggest that treatment of donor cells with TSA can facilitate their nucleus reprogramming by affecting the acetylated status of H4K8 and improving the in vitro development of buffalo SCNT embryos. The AcH4K8 status at the eight-cell stage can be used as an epigenetic marker for predicting the SCNT efficiency in buffalos.
    Full-text available · Article · Sep 2013 · Theriogenology
  • Hui Li · Xiangping Li · Qingyou Liu · [...] · Deshun Shi
    [Show abstract] [Hide abstract] ABSTRACT: Human interferon alpha 2b (IFNα-2b) is a pleiotropic cytokine used to treat various viral diseases and cancers. Conventionally, recombinant human IFNα-2b used in clinics was produced by prokaryotic expression system, which always lack of enough biological activity due to limitations on proper folding and post-translational modifications, so the eukaryotic expression system are becoming prevailing method for the production of recombinant proteins. In this study, human breast cancer cell Bcap-37 was firstly used as host for the expression of human IFNα-2b, with the expression vector pIRES2-IFN-EGFP, in which IFNα-2b gene is under the control of CMV promoter. The expression of recombinant IFNα-2b was detected by Western blot and ELISA. Results showed that the concentration of the secreted recombinant IFNα-2b in culture medium was 435.7 pg/mL/24 h. Biological activity of the recombinant IFNα-2b was assayed by detecting the expression of IFN-inducible genes, including MxA, OAS, PKR, and Caspase1 through QRT-PCR. Results demonstrated that recombinant IFNα-2b possess the biological activities. Compared to non-transgenic cells, the expression levels of the aforementioned four IFN-inducible genes were increased by 18.098-, 1.843-, 2.21-, and 3.066-folds, respectively. We got to a conclusion that the human breast cancer cell Bcap-37 could express bioactive recombinant IFNα-2b.
    Article · Aug 2013 · Applied biochemistry and biotechnology
  • Nan Li · Fenghua Lu · Peng Zhu · [...] · Deshun Shi
    [Show abstract] [Hide abstract] ABSTRACT: O-linked β -N-Ac ety lg luc os am me Glycosylation (O-GlcNAc) is one of the main types of glycosylation in mammalian cells while Glucosamine (GlcN) is an O-GlcNAc substrate. Thus, effects of GlcN on the embiyonic development, level of O-GlcNAc and related gene expression of buffalo embiyos were examined in this study. Buffalo zygotes derived from In Vitro Fertilization (IVF) were randomly allocated into culture in the medium supplemented with different concentration of GlcN (0, 1, 2 and 4 mM) during the different culture period (0-72, 72-172 and 0-172 h). When GlcN was added to the medium in the culture period of 0-72 h after IVF, addition of 2 mM GlcN resulted in more zygotes developing to blastocysts (26.1%) in comparison with control (14.3%), 1 mM (13.6%) and 4 mM (11.3%) groups (p<0.05). However, the blastocyst yield decreased gradually when GlcN was added to the medium during 72-172 h of culture and decreased significantly when the concentration of GlcN was arrived at 4 mM (3.1 vs. 14.2%, p<0.05). When GlcN was added to the medium in the whole culture period (0-172 h) there were no significant difference in either cleavage rate or blastocyst yield among the four groups (p>0.05). Immunofluorescence analysis revealed that addition of 2 mM GlcN to medium from 0-72 h after IVF resulted in a significant increase (p<0.05) in the O-GlcNAc level of embiyos at 2, 4, 8 cells and morula stage with the exception of blastocysts. QRT-PCR revealed that culture of zygotes with 2 mM GlcN in the culture period of 0-72 h after IVF resulted in a significant increase (p<0.05) in the expression of O-GlcNAc transferase gene in the embiyos at the 2, 4, 8 cells and morula stage and did not affect the expression of O-GlcNAc-selective N-acetyl β-D-glucosaminidase gene. These results indicate that appropriate concentration of GlcN can improve the development of buffalo embiyos and this action is stage dependent and mediated by O-GlcNAc transferase gene.
    Article · Jan 2013 · Journal of Animal and Veterinary Advances
  • Hui Li · Qingyou Liu · Kuiqing Cui · [...] · Deshun Shi
    [Show abstract] [Hide abstract] ABSTRACT: Interferon alpha 2b (IFNα-2b) is an important immune regulator widely used in clinic, for the treatment of chronic hepatitis, hairy cell leukemia, chronic myelogenous leukemia and multiple myeloma, etc. The clinically used IFNα-2b is generally produced by E.Coli, which lacks the post-translational O-glycosylation presents on naturally synthesized protein, and has a short serum half-life. In this study, a transgenic cassette pBCN-IFN-pA-CMV-EGFP was constructed, with a 5.2 kb beta-casein regulation fragment from Jersey cow and a 6×His tagged human Interferon alpha 2b (hIFNα-2b) gene fragment. By using pronuclear microinjection technique, transgenic mice were generated and the expression of IFNα-2b in the milk was assayed. The hIFNα-2b was correctly translated in milk of transgenic mice according to Western blot analysis. The expression level of hIFNα-2b was varied among the transgenic mice, and the highest one was about 29.71 μg/L. The recombinant protein exhibited biological activity in vitro by increasing the luminescence value and the MxA gene expression in established WISH cells, and the specific activity is approximately 2.8 × 10(7 )IU/mg. The expression of recombinant hIFNα-2b in mammary glands of transgenic mice constitutes an important step towards low-cost and full biological activity production of this protein drug in mammary gland bioreactor.
    Article · Jun 2012 · Transgenic Research
  • Yanfei Deng · Qingyou Liu · Chan Luo · [...] · Deshun Shi
    [Show abstract] [Hide abstract] ABSTRACT: Ectopically, expression of defined factors could reprogram mammalian somatic cells into induced pluripotent stem cells (iPSCs), which initiates a new strategy to obtain pluripotent stem cell lines. Attempts have been made to generate buffalo pluripotent stem cells by culturing primary germ cells or inner cell mass, but the efficiency is extremely low. Here, we report a successful method to reprogram buffalo fetal fibroblasts (BFFs) into pluripotent stem cells [buffalo induced pluripotent stem cell (biPSCs)] by transduction of buffalo defined factors (Oct4, Sox2, Klf4, and c-Myc) using retroviral vectors. The established biPSCs displayed typical morphological characteristics of pluripotent stem cells, normal karyotype, positive staining of alkaline phosphatase, and expressed pluripotent markers including Oct4, Sox2, Nanog, Lin28, E-Cadherin, SSEA-1, SSEA-4, TRA-1-81, STAT3, and FOXD3. They could form embryoid bodies (EBs) in vitro and teratomas after injecting into the nude BALB/C mice, and 3 germ layers were identified in the EBs and teratomas. Methylation assay revealed that the promoters of Oct4 and Nanog were hypomethylated in biPSCs compared with BFFs and pre-biPSCs, while the promoters of Sox2 and E-Cadherin were hypomethylated in both BFFs and biPSCs. Further, inhibiting p53 expression by coexpression of SV40 large T antigen and buffalo defined factors in BFFs or treating BFFs with p53 inhibitor pifithrin-a (PFT) could increase the efficiency of biPSCs generation up to 3-fold, and nuclear transfer embryos reconstructed with biPSCs could develop to blastocysts. These results indicate that BFFs can be reprogrammed into biPSCs by buffalo defined factors, and the generation efficiency of biPSCs can be increased by inhibition of p53 expression. These efforts will provide a feasible approach for investigating buffalo stem cell signal pathways, establishing buffalo stem cell lines, and producing genetic modification buffaloes in the future.
    Article · Mar 2012 · Stem cells and development
  • Qingyou Liu · Haiying Deng · Xiaoxi Zhang · [...] · Deshun Shi
    Article · Feb 2010 · Transgenic Research
  • Deshun Shi · Fenghua Lu · Yingming Wei · [...] · Qingyou Liu
    [Show abstract] [Hide abstract] ABSTRACT: Cloning of buffalos (Bubalus bubalis) through nuclear transfer is a potential alternative approach in genetic improvement of buffalos. However, to our knowledge, cloned offspring of buffalos derived from embryonic, fetal, or somatic cells have not yet been reported. Thus, factors affecting the nuclear transfer of buffalo somatic cells were examined, and the possibility of cloning buffalos was explored in the present study. Treatment of buffalo fibroblasts and granulosa cells with aphidicolin plus serum starvation resulted in more cells being arrested at the G0/G1 phase, the proportion of cells with DNA fragmentation being less, and the number of embryos derived from these cells that developed to blastocysts being greater. In addition, a difference was found in the development of embryos reconstructed with fetal fibroblasts from different individuals (P < 0.001). Forty-two blastocysts derived from granulosa cells and fetal fibroblasts were transferred into 21 recipient swamp buffalos, and 4 recipients were confirmed to be pregnant by rectal palpation on Day 60 of gestation. One recipient received two embryos from fetal fibroblasts aborted on Day 300 of gestation and delivered two female premature calves. Three recipients maintained pregnancy to term and delivered three female cloned calves after Days 338-349 of gestation. These results indicate that buffalo embryos derived from either fetal fibroblasts or granulosa cells can develop to the term of gestation and result in newborn calves.
    Article · Sep 2007 · Biology of Reproduction