Recombinant DNA applications Flashcards
Summary
Transgenesis
Recombinant proteins
molecular pharming
genetically modified organisms
gene vaccines
…
Transgenesis
- Introduction and expression of a gene of interest isolated from
one organism into another organism from a different species.
Transgenesis methods using naked DNA transfer
Transformation/Transfection
3 points
- This method uses competent cells induced chemically (CaCl2or Lithium-Acetate) to take in DNA.
- DNA is added to competent cells and let to enter the cells on ice.
- Usually a heat shock (42ºC) is required to complete DNA transfer.
Transgenesis methods using naked DNA transfer
Electroporation
3 points
- This method uses an electric pulse to drive
DNA into cells. - DNA is added to cells and placed in a cuvette
having electrodes on two sides. - An electric pulse is applied to the electrodes
using a pulse power supply.
Transgenesis methods using naked DNA transfer
Microinjection
3 points
This method involves the injection of naked DNA into cells using a pipette having a microscopic tip. It is mainly used for transforming animal cells (eggs). The operation is done under a microscope.
Transgenesis methods using naked DNA transfer
Bombardment
2 points
- This method uses a gene gun to transfer DNA coated on gold particles into host tissues.
- The gene gun uses compressed helium,
pulsed to project gold particles coated
with DNA into the cells.
Transgenesis methods using mediated DNA transfer
Liposome mediated transfer
DNA is “packaged” into liposomes which fuse to the plasma membrane releasing DNA inside the cell.
Transgenesis methods using mediated DNA transfer
Bacterial and viral mediated transfer
DNA is introduced in bacterial or viral strains which infect the target cells and transfer DNA into their genome.
Transgenesis methods using mediated DNA transfer
Selective breeding
This method is used to transfer DNA from one species to a close
species via successive crosses using a number of intermediates
rDNA applications: Production of recombinant proteins
3 points
- Production of proteins using an expression system transformed with
rDNA containing the coding sequence for the protein of interest. - Expression systems
Host cells together with compatible expression vectors allowing
high expression of recombinant proteins in these cells. - Hosts:
Bacteria: Escherichia coli
Rapid growth
Inducible system
Yeast: Saccharomyces ceriviseae
Eukaryotic protein folding system.
Good for producing functional recombinant eukaryotic proteins.
rDNA
Expression vectors
4 points
- Promoter
- ribosome binding site
- multiple cloning site
- termination of transcription
rDNA
Protein production systems
5 points
- Bacterial or yeast
culture
in small culture or Bioreactor (large optimal cultures - Cells are harvested
- Protein extraction
- Purification of the recombinant
protein - Applications
rDNA
Protein secretion: no extraction
- A signal peptide (SP) is usually required
for protein secretion outside the cells. - The nucleotide sequence encoding
the SP is added to the sequence encoding
the recombinant protein in the rDNA.
rDNA
3 Examples of therapeutic recombinant proteins
Insulin: hormone controlling glucose uptake by cells, diabetes.
Growth factor: used by young people having slow growth.
Coagulation factors: used in heamophiliac patients.
Molecular Pharming
- Production of pharmaceuticals in plants and animals.
- High production systems.
- low production costs
- Possibility for integrating pharmaceutical in food.
- Lower risk.
Pharming in plants
2 points
- rDNA encoding the therapeutic protein
Bombardment or electroporatin into cells
In vitro culture of transformed
cells on a selective medium
Regeneration of the transformed plants
- rDNA encoding the therapeutic protein
Insertion into a plasmid
Introduction of rDNA
into Agrobacterium
Infection of flowers with
Agrobacterium
Germination of seeds
on a selective medium
Transformed plants -> extraction of recombinant protein
Advantages of pharming in plants
5 points
- Many plant systems are easy to transform.
- Highly productive systems.
- Low maintenance and low cost.
- No possibility of disease transfer to humans.
- It is possible to produce pharmaceuticals in seeds: possibility of storage at room temperature conditions for a long time.
Pharming in Animals
5 points
- Hormonal treatment of donor ewes to induce ovulation followed by
insemination and collection of the fertilized eggs. - The human transgene is injected into the fertilized eggs.
- Transplantation of the eggs into a recipient.
- Selection for offspring producing the human protein in their milk.
- Selection for offspring producing the human protein in their milk.
Genetically Modified Organisms (GMOs)
Are organisms whose genome was modified via the insertion of rDNA to improve their phenotype.
Genetically Modified Plants
- GM crops represent about 30% (increasing) of the world total crop production including maize, soybean, rice, potato, cotton, squash.
- Genetic Modifications:
Resistance to pests: Bt maize.
Resistance to herbicides: maize.
Resistance to environmental stress: Maize, wheat, tomato.
Improved yield, post-harvest and nutritional qualities: rice, tomato.
Metabolic engineering
- Involves the optimization of genetic and regulatory processes within cells to increase the production of a certain substance.
- Usually requires the transfer of more than one gene intervening in the same or different metabolic pathways.
e.g. Golden rice:
Engineered rice that produces high levels (x23) of Vitamin A (βcarotene).
Genetically modified animals
- fish - faster growing salmon
2. Cows Increased Casein in Milk
DNA vaccines
5 points
- DNA encoding the antigen
- rDNA is inserted in a vector
- rDNA is transferred into the
animal to be immunized - The immunized animal produces the antigen protein
- The immunized animal produces antibodies against the antigen
- Protection against the disease for life
