Production of functional human nerve growth factor from the saliva of transgenic mice by using salivary glands as bioreactors

Zeng, Fang; Li, Zicong; Zhu, Qingchun; Dong, Rui; Zhao, Chengcheng; Li, Guoling; Li, Guo; Gao, Wenchao; Jiang, Gelong; Zheng, Enqin; Cai, Guangyan; Moisyadi, Stefan; Urschitz, Johann; Yang, Huaqiang; Liu, Dewu; Wu, Zhenfang

doi:10.1038/srep41270

Cited by 10 publications

(10 citation statements)

References 53 publications

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“…Transgenic plants producing antibodies may raise environmental concerns, whereas such problems are unlikely to be encountered with transgenic animals that are kept in enclosed areas [ 45 ]. Animal mammary gland bioreactor such as saliva [ 46 ] and other transgenic product such as blood [ 47 ] have proved to be a production platform for foreign proteins. Recombinant protein produced by transgenic animals is a cumbersome process and remains problematic in the application of this technology due to high cost and low expression efficiency [ 48 ].…”

Section: Discussionmentioning

confidence: 99%

Development of a Mucus Gland Bioreactor in Loach Paramisgurnus dabryanus

Zhou

Zhao

et al. 2021

IJMS

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Most currently available bioreactors have some defects in the expression, activity, or purification of target protein and peptide molecules, whereas the mucus gland of fish can overcome these defects to become a novel bioreactor for the biopharmaceutical industry. In this study, we have evaluated the practicability of developing a mucus gland bioreactor in loach (Paramisgurnus dabryanus). A transgenic construct pT2-krt8-IFN1 was obtained by subcloning the promoter of zebrafish keratin 8 gene and the type I interferon (IFN1) cDNA of grass carp into the SB transposon. The IFN1 expressed in CIK cells exhibited an antiviral activity against the replication of GCRV873 and activated two genes downstream of JAK-STAT signaling pathway. A transgenic loach line was then generated by microinjection of the pT2-krt8-IFN1 plasmids and in vitro synthesized capped SB11 mRNA. Southern blots indicated that a single copy of IFN1 gene was stably integrated into the genome of transgenic loach. The expression of grass carp IFN1 in transgenic loaches was detected with RT-PCR and Western blots. About 0.0825 µg of grass carp IFN1 was detected in 20 µL mucus from transgenic loaches. At a viral titer of 1 × 103 PFU/mL, plaque numbers on plates containing mucus from transgenic loaches reduced by 18% in comparison with those of the control, indicating that mucus of IFN1-transgenic loaches exhibited an antiviral activity. Thus, we have successfully created a mucus gland bioreactor that has great potential for the production of various proteins and peptides.

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Section: Discussionmentioning

confidence: 99%

Development of a Mucus Gland Bioreactor in Loach Paramisgurnus dabryanus

Zhou

Zhao

et al. 2021

IJMS

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“…The pmPSP-hNGF-EGFP plasmid, also named pmPSP-hNGF plasmid, was reported in a previous study [ 31 ]. pmPB was a kind gift from the Wellcome Trust Sanger Institute (Cambridgeshire, UK), which was constructed as previously described [ 32 ].…”

Section: Methodsmentioning

confidence: 99%

Genetically Engineered Pigs as Efficient Salivary Gland Bioreactors for Production of Therapeutically Valuable Human Nerve Growth Factor

Zeng

Liao

Kuang

et al. 2022

Cells

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Farm animal salivary glands hold great potential as efficient bioreactors for production of human therapeutic proteins. Nerve growth factor (NGF) is naturally expressed in animal salivary glands and has been approved for human clinical treatment. This study aims to employ transgenic (TG) pig salivary gland as bioreactors for efficient synthesis of human NGF (hNGF). hNGF-TG pigs were generated by cloning in combination with piggyBac transposon-mediated gene transfer. These hNGF-TG pigs specifically expressed hNGF protein in their salivary glands and secreted it at high levels into saliva. Surgical and nonsurgical approaches were developed to efficiently collect saliva from hNGF-TG pigs. hNGF protein was successfully purified from collected saliva and was verified to be biologically active. In an additional step, the double-transgenic pigs, where the endogenous porcine NGF (pNGF) gene was replaced by another copy of hNGF transgene, were created by cloning combined with CRISPR/Cas9-mediated homologous recombination. These double-transgenic pigs expressed hNGF but not pNGF, thus avoiding possible “contamination” of hNGF with pNGF protein during purification. In conclusion, TG pig salivary glands can be used as robust bioreactors for a large-scale synthesis of functional hNGF or other valuable proteins. This new animal pharming method will benefit both human health and biomedicine.

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“…A 890-bp DNA fragment containing the bacteria-inducible tracheal epithelial cell-specific bovine TAP promoter 49 – 51 and the 450-bp pig PG-1-coding sequences (GenBank Accession no: X79868.1) was synthesized by the GENEWIZ Company (Suzhou, China). This fragment was used to replace the PSP-hNGF fragment between the Age I and Asc I sites of the pmPSP-hNGF plasmid 52 , to generate the pTAP-PG-1 plasmid. The DNA sequences of pTAP-PG-1 plasmid were confirmed by sequencing.…”

Section: Methodsmentioning

confidence: 99%

Bacteria-induced expression of the pig-derived protegrin-1 transgene specifically in the respiratory tract of mice enhances resistance to airway bacterial infection

Zeng

Zhao

Xiao

et al. 2020

Sci Rep

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About 70% of all antibiotics produced in the world are used in the farm animal industry. The massive usage of antibiotics during farm animal production has caused rapid development of antibiotic resistance in bacteria, which poses a serious risk to human and livestock health when treating bacterial infections. Protegrin-1 (PG-1) is a potent antimicrobial peptide (AMP). It was initially identified in pig leukocytes with a broad-spectrum antibacterial and antiviral activity, and a low rate of inducing bacterial resistance. To develop a genetic approach for reducing the use of antibiotics in farm animal production, we produced transgenic mice carrying a bovine tracheal AMP gene promoter-controlled PG-1 transgene. The PG-1 transgene was specifically expressed in the respiratory tract of transgenic mice upon induction by bacterial infection. These PG-1 transgenic mice exhibited enhanced resistance to nasal bacterial infection as the transgenic mice showed a higher survival rate (79.17% VS. 34.78%), lower bacterial load and milder histological severity than their wild-type control littermates. The improved resistance to bacterial infection in the PG-1 transgenic mice could be resulted from the direct bacteria-killing activities of PG-1, and the immunomodulatory effects of PG-1 via stimulating interleukin 1 beta secretion. The present study provides a promising genetic strategy to prevent airway bacterial infections in farm animals by bacteria-inducible tissue-specific expression of PG-1 transgene. This approach may also be helpful for decreasing the possibility of inducing bacterial resistance during farm animal production.

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Production of functional human nerve growth factor from the saliva of transgenic mice by using salivary glands as bioreactors

Cited by 10 publications

References 53 publications

Development of a Mucus Gland Bioreactor in Loach Paramisgurnus dabryanus

Development of a Mucus Gland Bioreactor in Loach Paramisgurnus dabryanus

Genetically Engineered Pigs as Efficient Salivary Gland Bioreactors for Production of Therapeutically Valuable Human Nerve Growth Factor

Bacteria-induced expression of the pig-derived protegrin-1 transgene specifically in the respiratory tract of mice enhances resistance to airway bacterial infection

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