6 Introduction of DNA into living cells - uni-bayreuth.de Introduction of DNA into living cells 1....
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6 Introduction of DNA into living cells 1. Transformation and transfection 2. Protoplast transformation 3. Electroporation 4. In vitro packaging of λ DNA 5. Introduction of DNA into mammalian cells 6. Microinjection 7. The biolistic method
Transcript of 6 Introduction of DNA into living cells - uni-bayreuth.de Introduction of DNA into living cells 1....
6 Introduction of DNA into living cells
1. Transformation and transfection
2. Protoplast transformation
3. Electroporation
4. In vitro packaging of λ DNA
5. Introduction of DNA into mammalian cells
6. Microinjection
7. The biolistic method
6.1 Transformation and Transfection
Definition:
Transformation = Uptake of naked DNA by cells
Transfection = Uptake of naked viral DNA by cells
The Griffith Experiment (1928)
Pneumococci were capsulated, serum-resistant
The Griffith Experiment (1928)
Pneumococci were non-capsulated, serum-sensitive
The Griffith Experiment (1928)
The Griffith Experiment (1928)
Conclusion:
Some unknown compound is transferred from the heat-killed virulent to the non-virulent bacteria
Transforming principle
What is the chemical nature of this
transforming principle ?
Avery, McCarthy and McLeod (1944):
They fractionated the cytoplasmic content in protein and nucleic acids and checked both separately
The Avery Experiment (1944)
Conclusion:
DNA is the transforming principle!
and
Transformation has been discovered
Competence:
Physiological condition which allows bacteria to take up external DNA
Natural and induced competence
Only a few bacterial species become naturally competent:
Some species take up any DNA, e.g. B. subtilis
Other species take up only DNA carrying a particular signature (DNA sequence), e.g., Haemophilus influenzae
Transformation Protocol for E. coli
6.2 Protoplast Transformation
Principle:
Removal of the cell wall
6.3 Electroporation
Principle:
Exposure of the cells to a short electrical pulse
The Electroporation Procedure
6.4 In vitro Packaging of λ DNA
The Two-Strain System
The One-Strain System
In vitro Packaging of Recombinant λDNA: The Two-Strain System
no proheads no terminase
In vitro Packaging of Recombinant λDNA: The One-Strain System
The resident prophage does not contain a cos site →no packaging
6.5 Introduction of DNA Into
Mammalian Cells
Calcium phosphate method:
Precipitation of the DNA on the cell surface; uptake via endocytosis ?
DEAE (diethylaminoethyl) method:
DNA forms a complex with DEAE which is taken up via endocytosis
Liposome method:
Negatively charged phosphate groups of DNA bind to the positively charged surface of the liposomes
6.6 Microinjection
Direct injection of the DNA into the nucleus
Application:
Generation of transgenic animals
6.7 The Biolistic Method
Gene Delivery Microprojectiles Coated with DNA
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