Lectut btn-202-ppt-l25. introduction of dna into host cells
- 1. INTRODUCTION OF DNA INTO HOST CELLS 1 Lecture- 25
- 2. Introduction of biologically active recombinant DNA into host cells is a very important step in genetic engineering. TRANSFORMATION Introduction of DNA into a living cell TRANSFECTION (Infection by Transformation) Introduction of viral DNA into a living cell 2
- 3. METHODS FOR TRANSFORMATION/TRANSFECTION • BIOLOGICAL METHODS • DIRECT GENE TRANSFER by CHEMICAL AND BIOCHEMICAL MEANS • DIRECT GENE TRANSFER by PHYSICAL MEANS 3
- 4. BIOLOGICAL METHODS METHOD ORGANISM TRANSFORMED In vitro packaging and phage infection Bacteria Agrobacterium tumefaciens-mediated transformation Plants Plant-virus mediated transformation Plants Transposon-mediated transformation Insects Baculovirus-mediated transformation Insects Viral transduction Animals YAC transgenesis Animals 4
- 5. In vitro packaging and phage infection 5
- 7. DIRECT GENE TRANSFER BY PHYSICAL MEANS METHOD ORGANISM TRANSFORMED Microprojectile bombardment Plants Electroporation Plants, Animals Bacteria Microinjection Plants, Animals Silicon carbide fibers- mediated transformation Plants Ultrasonication Plants UV laser microbeam irradiation Plants 7
- 8. GENE GUN MEDIATED TRANSFORMATION 8
- 11. DIRECT GENE TRANSFER BY CHEMICAL MEANS METHOD ORGANISM TRANSFORMED CaCl2 / Heat shock Bacteria, Yeast Ca-DNA co-precipitation Plants, Animals Liposome packaging and fusion or Endocytosis Plants, Animals DEAE-dextran transfection Plants, Animals Poly cation-mediated DNA uptake Plants, Animals 11
- 12. PREPARATION OF COMPETENT CELLS AND TRANSFORMATION BY HEAT SHOCK 12
- 13. TRANSFORMATION EEFICIENCY •Number of colony forming units (cfus) produced by 1 microgram of plasmid DNA •TRANSFORMATION EEFICIENCY decreases with increase in size of DNA molecule. cfu = No. of bacterial colonies X dilution ratio X Original Transformation volume/plate volume 13
- 14. FACTORS AFFECTING TRANSFORMATION EEFICIENCY • GROWTH PERIOD • TEMPERATURE OF COMPETENT CELLS DURING PREPARATION, STORAGE AND TRANSFORMATION • CONCENTRATION OF CaCl2 • CULTURE MEDIUM 14