PLANT GENETIC ENGINEERING

1. PLANT GENETIC ENGINEERING AgustinaSetiawati

2. What are uses GM Plant? • Nutraceutical • Golden rice • Vitamin A enriched

3. The Golden Rice Story • Vitamin A deficiency is a major health problem • Causes blindness • Influences severity of diarrhea, measles • >100 million children suffer from the problem • For many countries, the infrastructure doesn’t exist • to deliver vitamin pills • Improved vitamin A content in widely consumed • crops an attractive alternative

4. IPP Geranylgeranyldiphosphate Phytoene synthase Phytoene Problem: Rice lacks these enzymes Phytoene desaturase ξ-carotene desaturase Lycopene Lycopene-beta-cyclase Normal Vitamin A “Deficient” Rice  -carotene (vitamin A precursor) -Carotene Pathway Problem in Plants

5. IPP Geranylgeranyl diphosphate Phytoene synthase Phytoene Vitamin A Pathway is complete and functional Phytoene desaturase ξ-carotene desaturase Lycopene Lycopene-beta-cyclase  -carotene (vitamin A precursor) Golden Rice The Golden Rice Solution -Carotene Pathway Genes Added Daffodil gene Single bacterial gene; performs both functions Daffodil gene

6. Antibody-producing tomato plant Nicholas Ewing California State University, Sacramento What are the Uses of GM Plants? • Bioreactors / Molecular farming • Therapeutic proteins • Human lactoferrin to treat iron deficiencies • Antibodies • Vaccine production • Antigen expression • HepC, HIV

7. Agrobacterium sp • Gram negative soil borne bacterium • Causes crown gall tumours • Mempunyaiplasmid Ti yang bisadipindahkankeseltanaman Crown-gall disease A.tumefaciens Hairy-root disease A.rhizogenes

8. Plasmid Ti

9. The limitation of Ti-plasmid • The production of phytohormon prevent being regenerated into mature plants • Ti-plasmid are large (200 to 800 kb) • Ti-plasmid does not replicate in E.coli Developing plant's cloning vector

10. T-DNA transfer to the plant genome

12. Cloning vector development based on T-DNA Co-integrative vector Binary cloning vector

13. The cointegrate cloning vector Cointegrate vector Disarmed Ti-plasmid Recombinant Ti-plasmid

14. The binary cloning vector

15. Co-integrative and binary vectors t-DNA LB RB Bacterial ORI Ampicillin resistance VIR genes PlasmidDNA Bacterial Chromosome Co-integrative Binary vector

16. TRANSFORMATION Metodetransformasipada GM plant: • Co-cultivation • Electroporation • Biolistic transformation – “Gene gun”

17. Co-cultivation Bacterial chromosome Ti Plasmid • Agrobacterium contains Ti plasmid recombinant • Co-cultivation of the Agrobacterium with plant pieces transfers the DNA Petri dish with leaf pieces plus Agrobacterium

19. General transformation protocol Transformation Agrobacterium sp Wash Co-cultivate (days) Transfer of t-DNA Inoculate (mins-hrs) (bacterial attachment) Sterile explants with dividing cells Recovery of transgenic plants Transfer to medium with bactericidal antibiotics (days) Kill off Agrobacterium Transfer to regeneration medium plus selective antibiotics Regeneration of transgenic plants Transfer to medium with bactericidal antibiotics plus selective antibiotics (months) Kill off Agrobacterium and select transgenic cells

20. Electroporation • Prinsip: pembukaanmembranpembentukanporiseltanamandenganmuatanlistrik • DNA in the surrounding solution can enter the cell through these pores and become incorporated into the cell’s nuclear genome through illegitimate recombination

21. Biolistic transformation – “Gene gun” • DNA is precipitated on the surface of heavy metal (gold; tungsten) particles • Loaded particles are driven into plant cells by high velocity gas propulsion (originally gunpowder; now helium) • Distance between discharge nozzle and tissue can be optimized, as can particle velocity • Target tissue must be regenerable

22. Once foreign DNA has been integrated into plant genomic DNA Have to be detected

23. A.Yuswanto, Fac. of Pharmacy, UGM

24. EDIBLE VACCINE • VIRUS-RESISTENT PLANT GM Plant is used for?

25. EDIBLE VACCINE Edible vaccines are vaccines produced in plants that can be administered directly through the ingestion of plant materials containing the vaccine. Eating the plant would then confer immunity against diseases. Edible vaccines produced by transgenic plants are attractive for many reasons. The cost associated with the production of the vaccine is low, especially since the vaccine can be ingested directly, and vaccine production can be rapidly up scaled should the need arises. Edible vaccine is likely to reach more individuals in developing countries. The first human clinical trial took place in 1997. Vaccine against the toxin from the bacteria E.coli was produced in potato. Ingestion of this transgenic potato resulted in satisfactory vaccinations and no adverse effects.

26. What exactly are“edible vaccines?” • Biopharmaceuticals • Plants or crops that produce human vaccines • The next generation of vaccines

27. Antibody production Plant as Bioreactors

28. VIRUS RESISTANT PLANT Cucumber mosaic virus

29. Binary vector containing either the protein-producing sense or the antisence of CuMV

31. Transgenic Animals

32. SOMATIC NUCLEAR CELL TRANSFER • Transgenic animal was constructed based on SNCT by Robert et al (1952)

33. DOLLY SHEEP, first succeed cloning

34. Uses for transgenic animals Gene pharming Food/Feed Xenotransplantation Industrial

35. Transgenic Animal • The foreign gene is constructed using recombinant DNA technology. • In addition to a structural gene, the DNA usually includes other sequences to enable it to be incorporated into the DNA of the host, and to be expressed correctly by the cells of the host.

36. Recombinant protein production in the milk of a transgenic sheep

37. Knock-in • A new gene is added (knocked in) by random insertion into the genome of the host organism. Your goal is to express that gene, but you don’t care where it ends up in the genome. • Circular plasmid construct DNA can break anywhere in its sequence and insert anywhere into the recipient cell genome. • Usually performed by microinjection into one of the two pronuclei of a newly fertilized egg. • Example: • Knock in a β-galactosidase gene driven by a promoter being tested for tissue specificity. • Watch where and when the β-galactosidase is expressed.

38. Transgenic animals “A little bit of this, and a little bit of that?”

39. Methods of producing transgenic animal • DNA microinjection • Embryonic stem (ES) cell transfer • Retroviral infection Source: A.Yuswanto, Fac. of Pharmacy, UGM 39

40. Microinjection Male pronuclei Pregnant mare serum gonadotropin follicular Human chorionic gonadotropin ovulation 40

41. A.Yuswanto, Fac. of Pharmacy, UGM 41

42. Retroviral infection Drawback ? Source: A.Yuswanto, Fac. of Pharmacy, UGM 42

43. Embryonic stem (ES) cell transfer Source: A.Yuswanto, Fac. of Pharmacy, UGM 43

46. Pronuclear injection. High efficiency for random knock-ins. How to analyse transgenic mice

47. Knock-out transgenic • A gene of the host organism is inactivated (knocked out) by insertion of a foreign sequence. • A mutant allele replaces the normal one by homologous recombination. • This is known as “targeted” insertion of a gene. • Targeting involves incorporating sequence identical to the target gene in the vector. • Successful homologous recombination is rare and must be • Selected and • Screened

48. KNOCKOUT MICE Normal (+) gene X Isolate gene X and insert it into vector. Inactivate the gene by inserting a marker gene that make cell resistent to antibiotic (e.g. puromycin) Genome Defective (-) Gene X Transfer vector with (-) gene X into ES cells (embryonic stem) VECTOR e.g.(NeoR) MARKER GENE