Genetic Engineering Methods

Genetic Engineering Methods

Outline Why do it? Research examples: poplar trees Plant gene transfer concepts and methods Getting genes ready for transfer (recombinant DNA/plasmids) Analysis of transgenic plants

Why do it? To create new, desired trait not in accessible gene pool To breed faster, more directly Dominant, rare alleles To amplify genetic diversity for particular genes/traits for breeding To breed with more direction, science basis To use plants as bioproduct factories for industry/medicine To promote biosafety for exotic/unsafe crop (domestication, sterility) In all cases, new traits/diversity feeds into breeding to check yield/stability of whole organism in the field: integrated health test

Native gene alteration example: Glutamine synthetase (GS) overexpression stimulates growth of poplars Hyper-expression of GS to stimulate ammonium nitrogen incorporation into proteins 3-year field trial (Spain) Increased nitrogen storage in stem Tree height up 41% Many examples, abiotic stress (F. Cánovas, U. Málaga, Spain)

Gene suppression example: Lignin modification improves pulping Energy and chemical costs of pulping great 70 million tons pulp (USA) ~$25 billion lignin removal (USA) Success in changing lignin amount and chemistry CAD - example Pulp yield (%) Pilate et al., INRA-France and UK 60 55 50 45 40 35 GE Control 15 17 19 21 23 25 27 Active alkali (%)

Exotic gene function example: Gene from bacterium makes trees more effective at bioremediation Thousands of square miles in U.S. mercury contaminated Neurotoxin, biomagnified mera transgenic cottonwood tolerates ~400 ppm mercury Volatilizing gene Wood-sequestering genes under development S. Merkle & R. Meagher, U Georgia

Biosynthetic pathway example: Modifying color/antioxidants Malonyl-CoA+ coumaroyl-coa P C2 Chalcone C2 C1 Vp1 (ABA) Flavanone R/B A1 Flavan-4-ol Dihydroflavonol Flavan-3,4-diol A1 PI R/B 3-Hydroxyanthocyanidine Bz1 Phobaphenes Anthocyanins

Biosafety example Sterile trees can dramatically reduce gene dispersal Variety of genetic mechanisms A tool to reduce risk of invasion by new exotic nursery/forestry species Invasive Douglas-firs in Argentina (B Bond)

Plant gene transfer concepts and methods

Summary of steps in plant genetic engineering

Antibiotic selection of transgenic cells Transformation Antibiotic selection Callus formation Shoot generation Root generation

DNA encodes both genes and signals for their control External Signal Cell Receptor Regulatory Elements Promoter Gene Terminator GO STOP Where When How Much TRANSCRIPTION mrna TRANSLATION Protein

Examples of Promoter:Gene Combos Promoter Gene 35 S-CAMV (virus) Bt (bacteria) Corn Stem Bt (bacteria) 35 S-CAMV Round-up Ready (bacteria) Tomato Fruit 5X Lycopene (tomato)

Reporter genes help visualize transgenic cells, promoter activity Any promoter GLO-FISH Fused to Any easily visualized gene Ubiqutious Fluorescence (JELLY FISH) FLORAL (POPLAR) GUS (BACTERIA)

Insertion of DNA into cells via biolistics ( gene gun )

Transgenic cassava via biolistics

Agrobacterium tumefaciens agent of crown gall disease

Agrobacterium is the method of choice for plant transformation A common soil pathogen that infects an large and taxonomically diverse range of plants A natural genetic engineer--gene transfer is essential to its pathogenic life style It transfers DNA into plant cells to cause gall formation, which provides a home and nutrition for it

Agrobacterium has a sophisticated gene transfer machinery Pathogenesis depends on presence of a very large plasmid, called the Ti (tumor inducing) plasmid, the source of its transferred genes (T-DNA) For biotechnology, the pathogenic genes are removed, replaced by useful genes

Agrobacterium life cycle

Agrobacterium transfer machinery

Agrobacterium Ti plasmid genes LB T - DNA auxin cytokinin opine RB vir genes ori opine catabolism Ti plasmid Right and left border (RB,LB) sequences are the only parts of T-DNA needed to enable transfer into plants removal of other T-DNA genes creates a disarmed plasmid

Disarmed and binary vector system Binary vectors live in E. coli too, and are used to modify & shuttle genes vir genes Disarmed Ti plasmid Agrobacterium Binary vector LB RB T- DNA Plant Cell

A sterilized paper punch is used to cut disks from plant leaves as the first step in genetic engineering. The cells on the edges of the disk are wounded in the process of cutting so they can receive a new gene from Agrobacterium tumefaciens.

Step-by-step view of poplar transformation

Summary of steps in regenerating transgenic plants using Agrobacterium

Getting genes ready for transfer

Restriction Enzymes cut DNA at specific DNA sequences (Alcamo. 1999. DNA Technology, 2nd Ed. Harcourt Press.)

Electrophoresis separates DNA fragments based on their length (Alcamo. 1999. DNA Technology, 2nd Ed. Harcourt Press.)

Construction of recombinant DNA molecules (Alcamo. 1999. DNA Technology, 2nd Ed. Harcourt Press.)

Boyer-Cohen Experiment, 1973 showed how genes could be cloned Plasmids are small, Circular DNA molecules that can replicate independently in a host cell. Foreign DNA inserted into plasmids can generate millions of copies of the inserted gene. (Alcamo. 1999. DNA Technology, 2nd Ed. Harcourt Press.)

Example of a map of binary plasmid used in plant transformation

Map of binary plasmid used in precommercial plant transformation

Analysis of transgenic plants

Example of repeated transgenes in a plant genome caused by transformation process

Southern (DNA) and northern (RNA) blots of transgenic cassava Produced via biolistics DNA gene presence RNA-gene expression

Expression analyses of Agrobacterium transformed tobacco Level of expression varies widely among independent gene transfer events

Many transgenic events need to be tested to find ones that are agronomically suitable Dozens to hundreds tested prior to commercial use Stable gene and trait expression (look for Mendelian inheritance like native gene) Single gene insertion for stability and simple breeding Desired level and pattern of expression (position effects)

Many transgenic events need to be tested to find ones that are agronomically suitable No deleterious effects on plant health/nearby genes (i.e., lack of somaclonal variation = unintended mutations) Introgression or insertion into other varieties for commercial use Regulation considers: Plant biochemistry, novel protein safety, allergenic potential, environmental impacts

The genome is a complex, messy, mutagenized, recombinant place! Natural transposable elements in the maize genome