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Fang R.,China Agricultural University | Peng Y.-Q.,China Agricultural University | Zheng M.,Beijing Genprotein Biotechnology Company | Meng Q.-Y.,China Agricultural University
Progress in Biochemistry and Biophysics | Year: 2012

Essential polyunsaturated fatty acids can not be synthesized in mammals due to the lack of Δ-12 and ω-3 fatty acid desaturases. Expressing ω-3 fatty acid desaturases in transgenic mammals could convert long chain n-6 polyunsaturated fatty acids to n-3 polyunsaturated fatty acids, significantly reducing the level of long chain n-6 polyunsaturated fatty acids. In this study, the muscle-specific transgenic mice expressed Caenorhabditis elegans FAT-1 and FAT-2 genes, which encode ω-3 and Δ-12 fatty acid desaturases respectively and human catalase hCAT gene, based on "self-cleaving" 2A peptides, and they were generated by microinjection. Fatty acids in skeletal muscle were analyzed by gas chromatography. The level of total n-3 polyunsaturated fatty acids in transgenic mice was 2.6-fold higher than that in wild type mice, while there was no significantly difference in the level of total n-6 polyunsaturated fatty acids between transgenic and wild type mice. But the skeletal muscle tissue of transgenic mice had a dramatically reduced ratio of n-6/n-3 fatty acids (P < 0.01). In addition, the expression level of human catalase was identified by Western blot, and the catalase activity of hCAT in skeletal muscle of transgenic mice was significantly higher than that of wild type mice (P< 0.01). Source


Cheng G.,China Agricultural University | Ding F.,Beijing Genprotein Biotechnology Company | Dai Y.,Beijing Genprotein Biotechnology Company | Li N.,China Agricultural University
African Journal of Biotechnology | Year: 2011

Targeting an exogenous gene into a favorable gene locus and for expression under endogenous regulators is an ideal method in mammary gland bioreactor research. For this purpose, a gene targeting vector was constructed to targeting the human lysozyme gene on bovine αs1-casein gene locus. In this case, the expression of human lysozyme could be regulated by the endogenous cis-element of αs1- casein gene in bovine mammary glands. In order to analyze the bioactivity of the vector, the targeting vector was stably transfected and randomly integrated into mouse mammary epithelial cells. Reversetranscription- polymerase chain reaction (RT-PCR) and western blot results showed that, the bovine αs1-casein promoter in the 5' arm was able to direct the efficient expression and secretion of human lysozyme in mammary epithelial cells. Turbidimetric assay showed that the antibacterial activity of lysozyme in transfected cells culture medium was 180 U/ml. To obtain the gene targeted cells line, bovine fetal fibroblasts were isolated and transfected with linear targeting vector (21.9 kb) using nucleofector device, which the transfection rate was about 25%. After seven rounds of independent cell transfection, a total of 8 × 10 7 cells were transfected, 118 colonies were expanded and analyzed by PCR, but none were found to be targeted. However, the targeted events were detected in the mixed cells which did not formed obvious colonies in five 10 cm dishes. Thus, these results indicate that, the 8.2 kb exogenous genes could be site specifically integrated into the transcriptionally silent αs1-casein gene locus in fibroblasts, but the unfavorable chromatin structure in such loci may have a disadvantage to targeted colonies formation in expansion stage. We suggest that minimizing the length of in vitro culture time and relax selection as soon as colonies become evident might prevent such loss of targeted cells. © 2011 Academic Journals. Source


Wei J.,China Agricultural University | Yang X.,PLA Fourth Military Medical University | Zheng M.,Beijing Genprotein Biotechnology Company | Wang M.,Beijing Genprotein Biotechnology Company | And 3 more authors.
Transgenic Research | Year: 2011

Biologically active recombinant monoclonal antibodies (mAbs) and their derivatives are in demand as therapeutic agents against a variety of cancers. The antibodies are generally produced by mammalian cell culture, but their production in the milk of transgenic animals would help meet the increasing demand. The mouse-human chimeric antibody chHAb18 has been proven to inhibit the invasion and metastasis of human hepatocellular carcinoma (HCC) cells by recognizing the HAb18G/CD147 molecule that is highly expressed on the surface of HCC tissue. Here, we report that transgenic mice generated by co-microinjection of two cassettes encoding the heavy and light chain genes of chHAb18 could highly express functional chHAb18 in their mammary glands. The expression level range of 1. 1-7.4 mg ml-1 was independent of transgenic copy number. Immunoassays demonstrated the ability and specificity of chHAb18 to bind purified antigen (i. e., HAb18G) or HCC cells. Recombinant chHAb18 from transgenic milk exhibited affinity almost equal to chHAb18 derived from CHO cells, and was 68% of that of the parental murine antibody, HAb18. In light of successful clinical application of HAb18, the chHAb18 expressed in mammary glands of transgenic mice constitutes an important step towards high-yield and scaled-up production of this antibody. © 2010 Springer Science+Business Media B.V. Source


Li Q.,China Agricultural University | Hu W.,China Agricultural University | Zhao J.,China Agricultural University | Wang J.,Beijing Genprotein Biotechnology Company | And 5 more authors.
Molecular Biology Reports | Year: 2014

Lactoferrin (LF) plays an important role in the body's immune system. However, the immunomodulatory effects of supplementation transgenic cow's milk containing recombinant human LF (rhLF) on the systemic and intestinal immune systems in infants remain unclear. Our laboratory has used genetic engineer to produce transgenic cow secreted rhLF. To assess the immune responses we took piglets as an animal model for infants. Eighteen piglets at 7 days of age were fed ordinary milk, 1:1 mix of ordinary and rhLF milk, or rhLF milk (LFM) for 30 days. The incidence of diarrhea in piglets in natural condition was observed. The protein abundances of immunoglobulin (Ig)G, IgA, IgM, IgE, histamine, interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12 interferon, tumor necrosis factor in the plasma, spleen or intestine were measured by enzyme-linked immunosorbent assay. Intestinal structure was assessed by hematoxylin and eosin. The mRNA levels of immune and allergy-related genes were measured by quantitative reverse transcription-polymerase chain reaction. The results showed that LFM-fed significantly reduced incidence of diarrhea, enhanced humoral immunity, T helper (Th) 1, and Th2 cell responses, improved the structure of the intestinal mucosal and did not induce food allergy. LFM increased mRNA levels of toll-like receptor 2 and nuclear factor-κB p65 and decreased that of FCεRI β. In conclusion, rhLF-enriched formula could improve systematic and intestinal immune responses and did not elicit food allergies in neonatal piglets. © 2014 Springer Science+Business Media Dordrecht. Source


Wang S.,China Agricultural University | Zhang K.,China Agricultural University | Ding F.,Beijing Genprotein Biotechnology Company | Zhao R.,Beijing Genprotein Biotechnology Company | And 6 more authors.
Journal of Biotechnology | Year: 2013

The PRNP gene encodes a cellular protein named prion, whose misfolded form has been implicated in a number of neuropathic diseases in mammals such as the Bovine Spongiform Encephalopathy (BSE) in cattle. BSE has brought devastating impact on the world economy and human health. Recently, several groups have performed the gene targeting strategy to disrupt the PRNP gene in bovine fibroblast cells and produce BSE-resistant cattle by somatic cell nuclear transfer (SCNT). However, the enrichment efficiency of the gene targeting vector was low. Here, we constructed a novel promoterless gene targeting vector to sequentially disrupt the PRNP gene in bovine fibroblast cells and generate gene targeted cattle by SCNT. The enrichment efficiency of the novel vector was 100% and 60%, respectively. After nuclear transfer, no significant difference was found in the rate of cleavage and blastocyst formation between the knockout and wild type cloned embryos. One PRNP+/- calf was born with no obvious abnormal development by now. Fusion RT-PCR and real-time PCR showed one allele of the PRNP gene was functionally disrupted, and the mRNA expression reduced dramatically in the PRNP+/- cattle. The reconstituted PRNP-/- embryos showed double alleles disruption, and no difference in the rate of cleavage and blastocyst formation. © 2012 Elsevier B.V. Source

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