Direct DNA transfer Introduce DNA into cells; assay expression immediately or select for permanently transformed cells. Techniques: Chemical induction Electroporation Particle bombardment (Biolistics) 1

Chemically-Induced Transformation Typically used on cells without walls There are multiple protocols Examples: Put DNA inside artificial membrane vesicles called liposomes, which will fuse with the plasma membrane of recipient cells, delivering the cargo. Bind DNA with polycations that neutralize the charged, sugar-PO4 backbone, and condense the DNA. Some cell types will endocytose this complex. Combine (1) and (2) 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 3

Particle Bombardment Less limitations than electroporation Can use on cells with walls Can transform organelles! Method: Precipitate DNA onto small tungsten or gold particles. Accelerate particles to high speeds and aim them at cells or tissues. Selective growth and regeneration of transgenic plants as described for Agro-mediated transformation. 4

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 5

An Air Rifle for a DNA Gun – Circa 1990 A.Thompson, Bob ?, and D. Herrin 6

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 7

The Helium Gas Gun – Circa 2000 8

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. 9