Making Transgenic Plants and Animals

1. Making Transgenic Plants and Animals • Why? • Study gene function and regulation • Making new organismic tools for other fields of research • Curing genetic diseases • Improving agriculture and related raw materials • New sources of bioengineered drugs (use plants instead of animals or bacteria)

2. Genetic Engineering of Plants • Must get DNA: • into the cells • integrated into the genome (unless using transient expression assays) • expressed (everywhere or controlled) • For (1) and (2), two main approaches for plants: • Agrobacterium - mediated gene transfer • Direct gene transfer • For (3), use promoter that will direct expression when and where wanted – may also require other modifications such as removing or replacing introns.

3. Agrobacterium - mediated Gene Transfer • Most common method of engineering dicots, but also used for monocots • Pioneered by J. Schell (Max-Planck Inst., Cologne) • Agrobacteria • soil bacteria, gram-negative, related to Rhizobia • species: tumefaciens- causes crown galls on many dicots rubi- causes small galls on a few dicots rhizogenes- hairy root disease radiobacter- avirulent

4. Crown galls caused by A. tumefaciens on nightshade. More about Galls: http://waynesword.palomar.edu/pljuly99.htm http://kaweahoaks.com/html/galls_ofthe_voaks.html

5. Agrobacterium tumefaciens • the species of choice for engineering dicot plants; monocots are generally resistant (but you can get around this) • some dicots more resistant than others (a genetic basis for this) • complex bacterium – genome has been sequenced; 4 chromosomes; ~ 5500 genes

6. Agrobacterium tumefaciens

7. Infection and tumorigenesis • Infection occurs at wound sites • Involves recognition and chemotaxis of the bacterium toward wounded cells • galls are “real tumors”, can be removed and will grow indefinitely without hormones • genetic information must be transferred to plant cells

8. Tumor characteristics • Synthesize a unique amino acid, called “opine” • octopine and nopaline - derived from arginine • agropine - derived from glutamate • Opine depends on the strain of A. tumefaciens • Opines are catabolized by the bacteria, which can use only the specific opine that it causes the plant to produce. • Has obvious advantages for the bacteria, what about the plant?

9. Elucidation of the TIP (tumor-inducing principle) • It was recognized early that virulent strains could be cured of virulence, and that cured strains could regain virulence when exposed to virulent strains; suggested an extra- chromosomal element. • Large plasmids were found in A. tumefaciens and their presence correlated with virulence: called tumor-inducing or Ti plasmids.

10. Ti Plasmid • Large (200-kb) • Conjugative • ~10% of plasmid transferred to plant cell after infection • Transferred DNA (called T-DNA) integrates semi-randomly into nuclear DNA • Ti plasmid also encodes: • enzymes involved in opine metabolism • proteins involved in mobilizing T-DNA (Vir genes)

11. T-DNA auxA auxB cyt ocs LB RB LB, RB – left and right borders (direct repeat) auxA + auxB – enzymes that produce auxin cyt – enzyme that produces cytokinin Ocs – octopine synthase, produces octopine These genes have typical eukaryotic expression signals!

12. auxAauxB • Tryptophan indoleacetamide  indoleacetic acid (auxin) • cyt • AMP + isopentenylpyrophosphate  isopentyl-AMP (a cytokinin) • Increased levels of these hormones stimulate cell division. • Explains uncontrolled growth of tumor.

13. Vir (virulent) genes • On the Ti plasmid • Transfer the T-DNA to plant cell • Acetosyringone (AS) (a flavonoid) released by wounded plant cells activates vir genes. • virA,B,C,D,E,F,G (7 complementation groups, but some have multiple ORFs), span about 30 kb of Ti plasmid.

14. Vir gene functions (cont.) • virA- transports AS into bacterium, activates virG post-translationally (by phosphoryl.) • virG- promotes transcription of other vir genes • virD2 - endonuclease/integrase that cuts T- DNA at the borders but only on one strand; attaches to the 5' end of the SS • virE2- binds SS of T-DNA & can form channels in artificial membranes • virE1 - chaperone for virE2 • virD2 & virE2 also have NLSs, gets T-DNA to the nucleus of plant cell • virB- operon of 11 proteins, gets T-DNA through bacterial membranes

15. From Covey & Grierson

16. Type IV Secretion Sys. • many pathogens, also used in conjugation • promiscuous • forms T-Pilus • B7-B10 span OM & IM • B7-B9 in OM interacts w/B8 & B10 of IM to form channel • 3 ATPases • D4 promotes specific transport • B2 can form filaments Gauthier, A. et al. (2003) J. Biol. Chem. 278:25273-25276

17. VirE2 may get DNA-protein complex across host PM Dumas et al., (2001), Proc. Natl. Acad. Sci. USA, 98:485

18. Monocots don't produce AS in response to wounding. • Important: Put any DNA between the LB and RB of T-DNA it will be transferred to plant cell! Engineering plants with Agrobacterium: Two problems had to be overcome: (1) Ti plasmids large, difficult to manipulate (2) couldn't regenerate plants from tumors

19. Binary vector system Strategy: 1. Move T-DNA onto a separate, small plasmid. 2. Remove aux and cyt genes. 3. Insert selectable marker (kanamycin resistance) gene in T-DNA. 4. Vir genes are retained on a separate plasmid. 5. Put foreign gene between T-DNA borders. 6. Co-transform Agrobacterium with both plasmids. 7. Infect plant with the transformed bacteria.

20. Binary vector system

21. 2 Common Transformation Protocols • Leaf-disc transformation - after selection and regeneration with tissue culture, get plants with the introduced gene in every cell • Floral Dip – does not require tissue culture. Reproductive tissue is transformed and the resulting seeds are screened for drug-resistant growth. (Clough and Bent (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal 16, 735–743)

22. Making a transgenic plant by leaf disc transformation with Agrobacterium. S.J. Clough, A.F. Bent (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal 16, 735–743.

23. Direct DNA transfer • Introduce naked DNA into cells; assay expression immediately or select for permanently transformed cells • DNA introduction: • Chemical • Electroporation • Particle bombardment (Biolistics)

24. Chemically-Induced Transformation • Usually use on cells without walls • Multiple protocols (examples): • Put DNA inside artificial membranes (liposomes), they will fuse with plasma membrane. • Bind DNA with polycations to neutralize charge, some cells endocytose the complex. • Combine (1) and (2)

25. Electroporation • Use on cells without walls (plant protoplasts or animal cells ) • High-voltage pulses cause pores to form transiently in cell membrane; DNA pulled in by electrophoresis or diffusion (?) • Drawback - its more cumbersome to regenerate plants from single protoplasts than from the tissue transformations with Agrobacterium

26. Particle Bombardment • Less limitations than electroporation • Can use on cells with walls, essentially any tissue • Can transform organelles! • Method: • Precipitate DNA onto small tungsten or gold particles. • Accelerate particles to high speeds at cells or tissues. • Selective growth and regeneration of transgenic plants as described for Agro-mediated transformation.

27. Original biolistic gun. A modified 22 caliber. DNA is bound to the microprojectiles, which impact the tissue or immobilized cells at high speeds. J. Sanford & T. Klein, 1988

28. An Air Rifle for a DNA Gun – Circa 1990 A.Thompson, Bob ?, and D. Herrin

29. Repairing an organellar gene: ~ 1 x 107 cells of a mutant of Chlamydomonas that had a deletion in the atpB gene for photosynthesis was bombarded with the intact atpB gene. Then, the cells were transferred to minimal medium so that only photosynthetically competent cells could grow. Control plate – cells were shot with tungsten particles without DNA

30. The Helium Gas Gun – Circa 2000

31. The Hand-Held Gas Gun Purpose: Introduce DNA into cells that are below the top surface layer of tissues (penetrate into lower layers of a tissue) One interesting use: Making DNA Vaccines in whole animals.

32. Transgenic Plants In Use or About to be on a Large Scale • Herbicide-resistant plants • Pest-resistant plants • Vaccine plants (just starting to be used)

33. Herbicide-resistant plants • Resistant to herbicide “Round-up” (Glyphosate) • Contain bacterial EPSP synthase • Advantages: better weed control, less tillage • soybeans, corn, rice, wheat

34. The function of EPSP synthase is to combine the substrate shikimate-3-phosphate (S3P) with phosphoenolpyruvate (PEP) to form 5-enolpyruvylshikimate-3-phosphate (EPSP).

35. Pest-resistant plants Cry5 • Resistant to certain insects • Lepidopterans, Coleopterans • Carry gene(s) for Bacillus thuringiensis (Bt) toxin • Toxin proteins produced as a parasporal crystal • Complex, composed of several proteins • Cry and Cyt genes • encoded on a plasmid • Advantage: less insecticide required, better yield • corn, cotton, potatoes A Transmission Electron Micrograph of negatively stained spores from Bt2-56 containing a filament (a), and a sac-like structure containing a spore (b) and parasporal body (c).

36. Insecticide Usage on Bt and non-Bt Cotton for 1999-2001

37. Vaccine plants • Pioneered by Charlie Arntzen • cheap vaccine-delivery system • use plants producing a pathogen protein (or DNA) to induce immunity • potatoes, bananas • being developed for a number of human and animal diseases, including measles, cholera, foot and mouth disease, and hepatitis B and C. • Four plant vaccines were successful in phase I clinical trials. C.J. Arntzen et al. (2005) Plant-derived Vaccines and Antibodies: Potential and Limitations. Vaccine 23, 1753-1756.

38. Concerns that have been raised about cultivating and consuming GM crops • They may be toxic or allergenic. • They may become established in the wild and outcompete other plants. • They may negatively affect insects or other organisms that use crops. • They may outcross to a nearby wild relative spreading the transgene into a wild population.

39. References on release of GM crops into the environment • Nap et al. (2003) Plant Journal 33, 1-18 • Focuses on current status and regulations • Conner et al. (2003) Plant Journal 33, 19-46 • Focuses on ecological risk assessment