Recombinant DNA applications

Question 1

Summary

Transgenesis

Recombinant proteins

molecular pharming

genetically modified organisms

gene vaccines

Answer 1

…

Question 2

Transgenesis

Answer 2

1. Introduction and expression of a gene of interest isolated from
   one organism into another organism from a different species.

Question 3

Transgenesis methods using naked DNA transfer

Transformation/Transfection

3 points

Answer 3

1. This method uses competent cells induced chemically (CaCl2or Lithium-Acetate) to take in DNA.
2. DNA is added to competent cells and let to enter the cells on ice.
3. Usually a heat shock (42ºC) is required to complete DNA transfer.

Question 4

Transgenesis methods using naked DNA transfer

Electroporation

3 points

Answer 4

1. This method uses an electric pulse to drive
   DNA into cells.
2. DNA is added to cells and placed in a cuvette
   having electrodes on two sides.
3. An electric pulse is applied to the electrodes
   using a pulse power supply.

Question 5

Transgenesis methods using naked DNA transfer

Microinjection

3 points

Answer 5

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.

Question 6

Transgenesis methods using naked DNA transfer

Bombardment

2 points

Answer 6

1. This method uses a gene gun to transfer DNA coated on gold particles into host tissues.
2. The gene gun uses compressed helium,
   pulsed to project gold particles coated
   with DNA into the cells.

Question 7

Transgenesis methods using mediated DNA transfer

Liposome mediated transfer

Answer 7

DNA is “packaged” into liposomes which fuse to the plasma membrane releasing DNA inside the cell.

Question 8

Transgenesis methods using mediated DNA transfer

Bacterial and viral mediated transfer

Answer 8

DNA is introduced in bacterial or viral strains which infect the target cells and transfer DNA into their genome.

Question 9

Transgenesis methods using mediated DNA transfer

Selective breeding

Answer 9

This method is used to transfer DNA from one species to a close
species via successive crosses using a number of intermediates

Question 10

rDNA applications: Production of recombinant proteins

3 points

Answer 10

1. Production of proteins using an expression system transformed with
   rDNA containing the coding sequence for the protein of interest.
2. Expression systems
   Host cells together with compatible expression vectors allowing
   high expression of recombinant proteins in these cells.
3. Hosts:

Bacteria: Escherichia coli
Rapid growth
Inducible system

Yeast: Saccharomyces ceriviseae
Eukaryotic protein folding system.
Good for producing functional recombinant eukaryotic proteins.

Question 11

rDNA
Expression vectors
4 points

Answer 11

1. Promoter
2. ribosome binding site
3. multiple cloning site
4. termination of transcription

Question 12

rDNA
Protein production systems
5 points

Answer 12

1. Bacterial or yeast
   culture
   in small culture or Bioreactor (large optimal cultures
2. Cells are harvested
3. Protein extraction
4. Purification of the recombinant
   protein
5. Applications

Question 13

rDNA

Protein secretion: no extraction

Answer 13

1. A signal peptide (SP) is usually required
   for protein secretion outside the cells.
2. The nucleotide sequence encoding
   the SP is added to the sequence encoding
   the recombinant protein in the rDNA.

Question 14

rDNA

3 Examples of therapeutic recombinant proteins

Answer 14

Insulin: hormone controlling glucose uptake by cells, diabetes.

Growth factor: used by young people having slow growth.

Coagulation factors: used in heamophiliac patients.

Question 15

Molecular Pharming

Answer 15

1. Production of pharmaceuticals in plants and animals.
2. High production systems.
3. low production costs
4. Possibility for integrating pharmaceutical in food.
5. Lower risk.

Question 16

Pharming in plants

2 points

Answer 16

1. 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

2. 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

Question 17

Advantages of pharming in plants

5 points

Answer 17

1. Many plant systems are easy to transform.
2. Highly productive systems.
3. Low maintenance and low cost.
4. No possibility of disease transfer to humans.
5. It is possible to produce pharmaceuticals in seeds: possibility of storage at room temperature conditions for a long time.

Question 18

Pharming in Animals

5 points

Answer 18

1. Hormonal treatment of donor ewes to induce ovulation followed by
   insemination and collection of the fertilized eggs.
2. The human transgene is injected into the fertilized eggs.
3. Transplantation of the eggs into a recipient.
4. Selection for offspring producing the human protein in their milk.
5. Selection for offspring producing the human protein in their milk.

Question 19

Genetically Modified Organisms (GMOs)

Answer 19

Are organisms whose genome was modified via the insertion of rDNA to improve their phenotype.

Question 20

Genetically Modified Plants

Answer 20

1. GM crops represent about 30% (increasing) of the world total crop production including maize, soybean, rice, potato, cotton, squash.
2. 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.

Question 21

Metabolic engineering

Answer 21

1. Involves the optimization of genetic and regulatory processes within cells to increase the production of a certain substance.
2. 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).

Question 22

Genetically modified animals

Answer 22

1. fish - faster growing salmon

2. Cows Increased Casein in Milk

Question 23

DNA vaccines

5 points

Answer 23

1. DNA encoding the antigen
2. rDNA is inserted in a vector
3. rDNA is transferred into the
   animal to be immunized
4. The immunized animal produces the antigen protein
5. The immunized animal produces antibodies against the antigen
6. Protection against the disease for life