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Production of Biologically Active Human GM-CSF Protein in Rice Seeds

Production of Biologically Active Human GM-CSF Protein in Rice Seeds. Katie Surckla. What is Human GM-CSF?. hGM -CSF stands for h uman G ranulocyte- M acrophage C olony S timulating F actor hGM -CSF is a cytokine which regulates the production and function of white blood cells.

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Production of Biologically Active Human GM-CSF Protein in Rice Seeds

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  1. Production of Biologically Active Human GM-CSF Protein in Rice Seeds Katie Surckla

  2. What is Human GM-CSF? • hGM-CSF stands for human Granulocyte-Macrophage Colony Stimulating Factor • hGM-CSF is a cytokine which regulates the production and function of white blood cells. • It is found in the body, but in very low concentrations • This protein is helpful for fighting a variety of infections.

  3. hGM-CSF clinical uses • Neutropenia • Pneumonia • Crohn’s fistulas • Diabetic foot infections • HIV-related opportunistic infections • Given to bone marrow transplant patients

  4. Therapuetic Recombinant Proteins • Majority of these proteins are produced in mammalian cells, or single celled organisms like yeast and bacteria and insects. • However, these methods are extremely expensive. • Ex.-Mammalian cell-based manufacturing facility can cost up to $250 million!

  5. Recombinant proteins • What else could we use these for? • Breast milk proteins • hGM-CSF is one of the many proteins that are looking to be added to breast milk by oral ingestion of recombinant proteins by the mother • This research is being done for mothers with HIV to resist passing on HIV to their children

  6. Major barriers • These methods can be challenging and hard to obtain high levels of expression of this protein • Public perceptions of these challenges • Getting approval to use land to field grow these transgene plants • Possible of inadvertent contamination of the food supply • Under stringent quality control and safety standards

  7. Why rice seeds? • Seeds are the most appealing target tissue • Seeds naturally store stable proteins for long periods of time • A large proportion of seed proteins belong to small sets of protein classes which helps in the purification steps. • Rice is also a popular weaning food for infants

  8. Rice endosperm cells • A 1.8 kb rice endosperm-specific glutelin promoter (Gt1) was used • Standard DNA cloning and DNA amplification techniques followed. Typical Rice Endosperm

  9. DNA cloning • Plasmid contains: • Gt1 promoter • 72 bp Gt1 signal sequence • *Transgene sugarcane production used particle gun bombardment while transgene rice seeds were produced in culture with a binary vector

  10. DNA cloning steps • 1) Digestion with NaeI enzyme • 2) Dephosphorylation • 3) hGM-CSF coding DNA sequence in BBG12 plasmid was amplified • 4) DNA sequence was phosphorylated

  11. DNA cloning steps • 5) The plasmid was involved in a ligation reaction with the Gt1 promoter, glutelin signal sequence and the GM-CSF DNA fragment. • 6) The transformed colony was identified • 7) The plasmid was cleaved with BamH1 and HincII enzymes

  12. DNA cloning steps • 8) This plasmid contained an EcoR1 site on the 5’ end and a NOS terminator sequence at the 3’ end. • 9) A HindIII site was added to the 5’ end of the Gt1 promoter • 10) This fragment was cloned into a binary vector, pCAMBIA 1301.

  13. Rice transformation, culture and growth • Agrobacterium strain LBA4404 was transformed with the binary vector pCAMBIA 1301 • Callus induction of rice seeds, callus selection and plant regeneration were performed and the plants were allowed to grow to about 8 inches. • The plants were then grown in pots in a controlled chamber at 28° C and about 50-60% humidity.

  14. Southern blot and PCR • The rice genomic DNA was then isolated and purified • Southern blot-approximately 10 μg of rice DNA was digested and separated on 0.8% agarose gel, denatured then transferred to a nylon membrane. • The membrane was probed with 32P labeled fragment Fig. 1-Southern Blot analysis on genomic DNA from rice plants. Lanes 1 and 2: positive control as HindIII insert released from the construct. Lanes 3-8: HindIII-cleaved genomic DNA from independent transgenic rice plants.

  15. Western blotting • Clarified seed extracts were separated on 15% SDS polyacrylamide gels. • The proteins were transferred to PVDF membranes and treated with a blocking buffer solution. • Protein bands were visualized using the NBT/BCIP substrates.

  16. Western blot results • Lanes 1-2: E. coli derived GM-CSF at 2 different concentrations. • Lanes 3-4: Non-transgenic plants • Lane M: Prestained molecular weight marker • Lanes 5-7: Transgenic rice plants of different concentrations

  17. Gt1 promoter • The 1.8 kb Gt1 glutelin promoter from rice was used to control the expression of the hGM-CSF mature coding sequence. • The glutelin signal sequence was ligated in-frame with the coding sequence of GM-CSF. • Finally cloned into a binary vector, pCAMBIA 1301 which was then transferred to the competent LBA4404 strain of Agrobacterium

  18. Integration of GM-CSF DNA in the rice genome • The Agrobacterium cells were then used to transform vigorously growing rice calli. • 6 transgenic plants regenerated from calli • PCR was used to verify the presence of the hGM-CSF sequence • Furthermore, the DNA was digested with HindIII to verify the integration into the rice genome

  19. Detection in rice seeds • An hGM-CSF specific ELISA assay was used • 1.2% tsp for plant #1 (28 μg/ml GM-CSF) • 1.3% tsp for plant #2 (28 μg/ml GM-CSF) • Western blot also showed bands at 18 kDa (weight of GM-CSF in the non-glycosylated form)

  20. Biological activity of hGM-CSF • Was tested using a human cell line, TF-1 • This medium only grows in the presence of hGM-CSF or other growth factors • Assay medium alone (without GM-CSF) did not support proliferation of TF-1 cells • With the GM-CSF, TF-1 cells proliferated

  21. Results of sugarcane and rice • Sugarcane • Out of 34 tested plants, 22 showed unique hybridization patterns • Average tsp ~0.1% • Rice • Out of 6 tested plants, 2 showed unique hybridization • Average tsp ~1.3%

  22. Factors limiting sugarcan e • Transgene silencing (Post-transcriptional gene silencing, PTGS) • Rapid mRNA turnover due to specific mRNA-destabilizing elements

  23. Conclusions • Overall, the rice plants were found to have 1.3% tsp • This is 4-fold higher than the reported expression level in the seed of tobacco! • Also, we can achieve even higher levels of protein by employing a larger version of the Gt1 promoter

  24. Future plans • Hopefully we can successfully amplify this protein in sugarcane or rice seeds • Oral ingestion of hGM-CSF is not expected to have an immune response since these plants are typically ingested • Hopefully we can find a cheaper cost of production of this protein for clinical use • Help with humanizing breast milk for third world countries

  25. References • Sardana, Ravinder. et. al. 2007. Biologically Active Human GM-CSF Produced in the Seeds of Transgenic Rice Plants. Transgenic Research. 16: 713-721. • Wang, Ming-Li. et. al. 2004. Production of Biologically Active GM-CSF in sugarcane: a secure biofactory. Transgenic Research. 14: 167-178. • Blais, David R. et. al. 2007.Humanizing infant milk formula to decrease postnatal HIV transmission. TRENDS in Biotechnology. 25(9): 376-384.

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