Katie Surckla. hGM-CSF stands for human Granulocyte- Macrophage Colony Stimulating Factor hGM-CSF...
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Transcript of Katie Surckla. hGM-CSF stands for human Granulocyte- Macrophage Colony Stimulating Factor hGM-CSF...
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Production of Biologically Active Human GM-CSF Protein in Rice Seeds
Katie Surckla
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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.
What is Human GM-CSF?
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Neutropenia
Pneumonia
Crohn’s fistulas
Diabetic foot infections
HIV-related opportunistic infections
Given to bone marrow transplant patients
hGM-CSF clinical uses
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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!
Therapuetic Recombinant Proteins
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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
Recombinant proteins
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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
Major barriers
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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
Why rice seeds?
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Rice endosperm cells
Typical Rice Endosperm
A 1.8 kb rice endosperm-specific glutelin promoter (Gt1) was used
Standard DNA cloning and DNA amplification techniques followed.
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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
DNA cloning
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1) Digestion with NaeI enzyme
2) Dephosphorylation
3) hGM-CSF coding DNA sequence in BBG12 plasmid was amplified
4) DNA sequence was phosphorylated
DNA cloning steps
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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
DNA cloning steps
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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.
DNA cloning steps
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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.
Rice transformation, culture and growth
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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
Southern blot and PCR
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.
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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.
Western blotting
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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
Western blot results
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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
Gt1 promoter
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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
Integration of GM-CSF DNA in the rice genome
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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)
Detection in rice seeds
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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
Biological activity of hGM-CSF
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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%
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Transgene silencing (Post-transcriptional gene silencing, PTGS)
Rapid mRNA turnover due to specific mRNA-destabilizing elements
Factors limiting sugarcane
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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
Conclusions
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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
Future plans
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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.
References