Genetic Engineering Methods - Oregon State University

1 Genetic Engineering MethodsOutline Why do it? Research examples: poplar trees Plant gene transfer concepts and Methods Getting genes ready for transfer (recombinant DNA/plasmids) Analysis of transgenic plantsWhy 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 bioproductfactories 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 testNative gene alteration example:Glutamine synthetase(GS) over-expression 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.)

2 C novas, U. M laga,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-example35404550556015171921232527 Pulpyield(%)GEControlPilate et al., INRA-France and UKActive alkali (%)Exotic gene function example:Gene from bacterium makes trees more effective at bioremediation Thousands of square miles in mercury contaminated Neurotoxin, biomagnified merAtransgenic cottonwood tolerates ~400 ppmmercury Volatilizing gene Wood-sequestering genes under developmentS. Merkle& R. Meagher, U GeorgiaMalonyl-CoA+coumaroyl-CoABiosynth etic pathway example:Modifying color/antioxidantsAnthocyaninsPhobaphene sChalconeFlavan-4-olFlavanoneDihydroflav onolA1Bz1A1C2C2 PFlavan-3,4-diol3-Hydroxy-anthocyanidine Vp1C1(ABA)R/BPIR/BBiosafety exampleSterile trees can dramatically reduce gene dispersal Variety of Genetic mechanisms A tool to reduce risk of invasion by new exotic nursery/forestry speciesInvasive Douglas-firs in Argentina (B Bond)Plant gene transfer concepts and Methods Summary of steps in plant Genetic engineeringAntibioticselectionTransforma tionCallusformationShootgenerationRootge nerationAntibiotic selection of transgenic cellsTRANSCRIPTIONTRANSLATIONR egulatory ElementsWhenHowMuchPromoterGeneTerminato rSTOPGOCell ReceptorDNA encodes both genes and signals for their controlExternal SignalWheremRNAP roteinExamples of Promoter.

3 Gene CombosPromoterGene35S-CAMV (virus) Bt (bacteria)Corn StemBt (bacteria)Round-upReady (bacteria)35S-CAMVT omato Fruit 5 XLycopene(tomato)Reporter genes help visualize transgenic cells, promoter activityAny promoterAny easily visualized geneFused toUbiqutiousFluorescence(JELLY FISH)GLO-FISHFLORAL(POPLAR)GUS(BACTERIA) Insertion of DNA into cells via biolistics( gene gun )Transgenic cassava via biolisticsAgrobacteriumtumefaciensagent of crown gall disease 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 itAgrobacteriumis the method of choice for plant transformationAgrobacteriumhas 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 genesAgrobacteriumlife cycleAgrobacteriumtransfer machineryAgrobacteriumTi plasmid genesLBRB auxincytokininopinevirgenesoriopine catabolismT -DNATi plasmidRight and left border (RB,LB)

4 Sequences are the only parts of T-DNA needed to enable transfer into plants removal of other T-DNA genes creates a disarmedplasmidDisarmed and binary vector systemBinary vectors live in E. coli too, and are used to modify & shuttle genes AgrobacteriumDisarmedTi plasmidBinary vectorRBLB virgenesT-DNAP lant CellA sterilized paper punch is used to cut disks from plant leaves as the first step in Genetic cells on the edges of the disk are wounded in the process of cutting so they can receive a new gene from view of poplar transformationSummary of steps in regenerating transgenic plants using AgrobacteriumGetting genes ready for transferRestriction 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.)

5 1999. DNA Technology, 2nd Ed. Harcourt Press.)Boyer-Cohen Experiment, 1973 showed how genes could be clonedPlasmids are small, Circular DNA molecules that can replicate independently in a host 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 transformationMap of binary plasmid used in pre-commercial plant transformationAnalysis of transgenic plantsExample of repeated transgenes in a plant genome caused by transformation processSouthern (DNA) and northern (RNA) blots of transgenic cassavaProduced via biolisticsDNA gene presence RNA-gene expressionExpression analyses of Agrobacteriumtransformed tobaccoLevel of expression varies widely among independent gene transfer eventsMany transgenic events need to be tested to find ones that are agronomicallysuitable Dozens to hundreds tested prior to commercial use Stable gene and trait expression (look for Mendelianinheritance like native gene) Single gene insertion for stability and simple breeding Desired level and pattern of expression (position effects)

6 No deleterious effects on plant health/nearby genes ( , lack of somaclonalvariation = unintended mutations) Introgression or insertion into other varieties for commercial use Regulation considers: Plant biochemistry, novel protein safety, allergenic potential, environmental impactsMany transgenic events need to be tested to find ones that are agronomicallysuitableThe genome is a complex, messy, mutagenized, recombinant place!Natural transposable elements in the maize genom