This document discusses various methods for introducing DNA into host cells, which is an important step in genetic engineering. It describes transformation, which is introducing DNA into living cells, and transfection, which is introducing viral DNA into living cells. The document then outlines several biological, chemical, and physical methods for transformation and transfection in bacteria, plants, insects, and animal cells. These include techniques like bacterial infection with bacteriophage, Agrobacterium-mediated plant transformation, microprojectile bombardment, electroporation, microinjection, and calcium phosphate transfection. It also discusses factors that affect transformation efficiency.

1. INTRODUCTION OF DNA
INTO HOST CELLS
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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
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3. METHODS FOR
TRANSFORMATION/TRANSFECTION
• BIOLOGICAL METHODS
• DIRECT GENE TRANSFER by
CHEMICAL AND BIOCHEMICAL MEANS
• DIRECT GENE TRANSFER by
PHYSICAL MEANS
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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
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5. In vitro packaging and phage infection
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6. Agrobacterium MEDIATED TRANSFORMATION
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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
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8. GENE GUN MEDIATED TRANSFORMATION
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9. ELECTROPORATION MACHINE MECHANISM
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10. MICROINJECTION
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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
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12. PREPARATION OF COMPETENT CELLS AND
TRANSFORMATION BY HEAT SHOCK
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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
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14. FACTORS AFFECTING TRANSFORMATION EEFICIENCY
• GROWTH PERIOD
• TEMPERATURE OF COMPETENT CELLS DURING
PREPARATION, STORAGE AND TRANSFORMATION
• CONCENTRATION OF CaCl2
• CULTURE MEDIUM
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