6.4 -Recombinant DNA for Transgenic Organism

Question 1

Gene cloning

Answer 1

Target gene + vector. Then replicating into a host cell.
The plasmid and gene is made into recombinant DNA and is inserted into the organism/cell that needs that gene.

Donor DNA is isolated by

• Restriction endonuclease enzyme - specific restriction site, producing sticky or blunt ends
• Reverse transcriptase enzyme - synthesises complementary DNA using its mRNA.

Selection of a suitable vector

• Used to clone genes, that once inserted does not change the effect of the inserted gene.
• It must be self-replicating inside host cell
• Possess the same restriction sites (so the same RE enzyme is used)
• Introduction of donor DNA fragment must not interfere with replication property of the vector.
• Must possess a marker gene so that it can used for later identification of recombinant cell. E.g. nonfunctional lacZ gene: if it were functional it would break down X-gall, making a blue colour in the colonies.

Incorporation of DNA fragment into the vector

• The plasmid vector is cut open by the same restriction endonuclease enzyme.
• Ligation process: fragments form a recombinant vector in the mixture.

Transformation of recombinant vector into a suitable host i.e bacterial cell such as e.coli

Isolation of recombinant host cell

• The colonies are grown in a culture media with ampicillin and X-gall present.
• This is to check the success of the recombinant plasmid - some functions that rejects the gene.
• Only bacteria with resistance to ampicillin (recombinant plasmid) will survive
• Recombinant colonies would be white, not blue (non-functional lacZ).

Question 2

Purpose of Ligase

Answer 2

The same as in DNA replication - to create a seamless DNA moleucle.

• DNA replication: to remove nicks from removed RNA primers, Okazaki fragments
• Gene cloning: join target gene and plasmid vector.

Question 3

Annealing process

Answer 3

In recombinant DNA technologies:

• Human gene and plasmid join together via base pairings.

In PCR:

• Primers to form hydrogen bonds with their complementary sequences on the single-stranded template DNA.

Question 4

Transgenic species

Answer 4

The genetic transformation must occur in the organism’s germ-line tissue (egg, sperm) so that the gene is passed to the next generation. These transgenic species have genes from another organism inserted into their genome.

Question 5

Transgenic crops

Answer 5

Transformed A. tumefaciens is cultured with plant cells → the recombinant plasmid can move from the bacterium into plant cells and be directed into the chromosomes of plants.
E.g. GM canola, herbicide tolerant.

Question 6

Pharming

Answer 6

Genetic engineering to insert genes that code for useful pharmaceuticals into host animals or plants.

• E.g. ATryn goats, Tracy the Sheep although this treatment of cystic fibrosis caused breathing problems and was discontinued.

Question 7

Knock out mice

Answer 7

“Knock out”: existing gene is replaced or disrupted with an artificial piece of DNA.
This loss of gene activity causes changes in a mouse’s phenotype (appearance, behaviour, observable physical and biochemical characteristics)

• Valuable clues on what the gene normally does, as humans share many genes with mice.
• Deeper understanding on how a similar gene could cause/contribute to disease in humans.
• These mice have modelled for obesity, heart disease, diabetes, arthritis, substance abuse, anxiety, aging, Parkinson disease.
• Also offer a biological context in which drugs and other therapies can be developed and tested.

Question 8

Vaccine research

Answer 8

Antigen is extracted - cheap vaccine with few side effects (not the actual virus).

This is through recombinant technologies:

• E.g. Hep. B virus
• Target DNA inserted into a bacterial plasmid, combined with yeast cell.
• Fermentation, antigen is extracted.

Question 9

Xenotransplantation

Answer 9

Transplantation of tissues between individuals of different species.

• Alleviate pressure + provide necessary resouce as the demand for organ transplants vastly outweigh the number of available organs at any time.
• Transgenic species, such as pigs, are engineered to have matching surface marker with humans to avoid organ rejection. Organs from pigs, such as kidneys and livers, that have been engineered to have surface markers matching those of humans, can then be harvested and transplanted into human patients

Question 10

Creating a transgenic organism

Answer 10

Animals:

Virus:

Agrobacterium: