Direct DNA transfer

• Introduce DNA into cells; assay expression immediately or select for permanently transformed cells.

• Techniques: 1. Chemical induction 2. Electroporation 3. Particle bombardment (Biolistics)

Chemically-Induced Transformation

• • Typically used on cells without walls There are multiple protocols Examples: 1. Put DNA inside artificial membrane vesicles called liposomes, which will fuse with the plasma membrane of recipient cells, delivering the cargo.

2. Bind DNA with polycations that neutralize the charged, sugar-PO 4 backbone, and condense the DNA. Some cell types will endocytose this complex.

3. Combine (1) and (2)

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 and diffusion. • Drawback - its more cumbersome to regenerate plants from single protoplasts than from the tissue transformations with

Agrobacterium

Particle Bombardment

• • • Less limitations than electroporation Can use on cells with walls Can transform organelles!

• Method: 1. Precipitate DNA onto small tungsten or gold particles.

2. Accelerate particles to high speeds and aim them at cells or tissues.

3. Selective growth and regeneration of transgenic plants as described for Agro-mediated transformation.

Original 22-caliber biolistic gun DNA is bound to the microprojectiles, which impact the tissue or immobilized cells at high speeds.

J. Sanford & T. Klein, 1988

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

Repairing an organellar gene

: ~ 1 x 10 7 cells of a mutant of

Chlamydomonas

that had a deletion in the

atpB

gene for photosynthesis was bombarded with the intact gene. Then, the cells were transferred to minimal medium so that only photosynthetically competent cells could grow.

atpB

Control plate – cells were shot with tungsten particles without DNA

The Helium Gas Gun – Circa 2000

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.

Transgenic Plants In Use on a Large Scale

• Herbicide-resistant plants • Pest-resistant plants • Vaccine plants (just starting to be used)

Herbicide-resistant plants

• Resistant to herbicide “Round-up” (glyphosate), which inhibits EPSP synthase.

• GEngineered plants contain a bacterial EPSP synthase, which is not inhibited by glyphosate.

• Advantages: better weed control, less tillage • In use: soybeans (dicot), corn, rice, wheat

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

Pest-resistant plants

• Resistant to certain insects – Lepidopterans, Coleopterans • Carry gene(s) for

Bacillus thuringiensis

(

Bt

) toxin • Toxin proteins produced as a parasporal crystal – crystalline material has several proteins –

Cry

and

Cyt

genes – encoded on a plasmid • Advantage: less insecticide required, better yield • corn, cotton, potatoes Cry5 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).

Insecticide Usage on Bt and non-Bt Crops for 1999-2001

Vaccine plants

• pioneered by Charlie Arntzen • cheap vaccine-delivery system • plant produces protein(s) or DNA from the human pathogen, and immunity is induced via food – 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.

Concerns that have been raised about cultivating/consuming GM crops (or GMOs)

1. They may be toxic or allergenic.

2. They may become established in the wild and outcompete other plants.

3. They may negatively affect insects or other organisms that use crops. 4. They may outcross to a nearby wild relative spreading the

trans

gene into a wild population.

References on regulation and eco risk assessment vis à-vis the cultivation of GM crops

• 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 • GM Crops: A World View.

Science

, April 2008.