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Engineering Camelina sativa Seeds as a Green Bioreactor for the Production of Affordable Human Pro-insulin that Demonstrates Anti-diabetic Efficacy in Rats

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Abstract

The current production of recombinant insulin via fermenter-based platforms (Escherichia coli and yeast) could not fulfill its fast-growing commercial demands, thus leading to a great interest in its sustainable large-scale production at low cost using a plant-based system. In the present study, Agrobacterium tumefaciens-mediated nuclear stable genetic transformation of an industrial oilseed crop, Camelina sativa, to express pro-insulin (with three furin endoprotease cleavage sites) fused with cholera toxin B subunit (CTB) in their seeds was successfully achieved for the first time. The bar gene was used as a selectable marker for selecting transformants and producing herbicide-resistant camelina plants. The transformation process involved the infiltration of camelina inflorescences (at flower buds with partially opened flowers) with A. tumefaciens and harvesting the seeds (T0) at maturity. The T0 seeds were raised into the putative T1 plants sprayed with Basta herbicide (0.03%, v/v), and the survived green transformed plants tested positive for pro-insulin and bar genes. A transformation frequency of 6.96% was obtained. The integration and copy number of the pro-insulin transgene and its expression at RNA and protein levels were confirmed in T1 plants using Southern hybridization, semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction (sqPCR), and quantitative real-time Time PCR (qPCR) and western blot analysis, respectively. Enzyme-linked immunosorbent Assay (ELISA) quantified the amount of expressed pro-insulin protein, and its anti-diabetic efficacy was validated in diabetic rats on oral feeding. Transgenic plants integrated the pro-insulin gene into their genomes and produced a maximum of 197 µg/100 mg of pro-insulin (0.804% of TSP) that had anti-diabetic efficacy in rats.

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Acknowledgements

PKJ and SB are thankful to the University Grants Commission, New Delhi, for the award of the Basic Science Research-Faculty Fellowship and Senior Research Fellowship, respectively and to Prof. Edgar Cahoon, University of Nebraska-Lincoln, USA for pBinGy1bar. No special funding for this work was available.

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Authors and Affiliations

  1. Centre for Biotechnology, M. D. University, Rohtak, 124001, India

    Sapna Bhoria, Darshna Chaudhary & Pawan K. Jaiwal

  2. Department of Zoology, M. D. University, Rohtak, 124001, India

    Priyanka Saini & Ranjana Jaiwal

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  1. Sapna Bhoria
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  2. Priyanka Saini
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  3. Darshna Chaudhary
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  4. Ranjana Jaiwal
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Contributions

SB contributed toward methodology, investigation, analysis, and writing-original draft. PS contributed toward interpretation of data. DC contributed toward project administration, resources, writing review, and editing. RJ contributed toward conceptualization, study design, writing review, and editing. PKJ contributed toward conceptualization, supervision, writing review, and editing.

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Correspondence to Pawan K. Jaiwal.

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The use of small animals in the present study was approved by the Institutional Animal Ethical Committee (IAEC) of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals), M. D. University, Rohtak-124001(India).

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Bhoria, S., Saini, P., Chaudhary, D. et al. Engineering Camelina sativa Seeds as a Green Bioreactor for the Production of Affordable Human Pro-insulin that Demonstrates Anti-diabetic Efficacy in Rats. Mol Biotechnol (2024). https://doi.org/10.1007/s12033-024-01068-y

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