1. Katie Surckla

2.  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.  Neutropenia  Pneumonia  Crohn’s fistulas  Diabetic foot infections  HIV-related opportunistic infections  Given to bone marrow transplant patients

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

9.  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.  1) Digestion with NaeI enzyme  2) Dephosphorylation  3) hGM-CSF coding DNA sequence in BBG12 plasmid was amplified  4) DNA sequence was phosphorylated

11.  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.  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.  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.  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 32 P 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.  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.  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.  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.  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.  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.  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.  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.  Transgene silencing (Post-transcriptional gene silencing, PTGS)  Rapid mRNA turnover due to specific mRNA- destabilizing elements

23.  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.  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.  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.