Transgenic models of human disease

Question 1

What can a mutation in a single gene cause?

Answer 1

A disease

Question 2

What is homologous recombination?

Answer 2

The swapping round of genetic material

Question 3

What are transgenic models of human disease?

Answer 3

A mutant form of a gene (with a selection marker) is inserted into the nucleus of an embryonic stem cell (via a vector), and then allowed to grow into a whole animal

Question 4

What is the role of the selection marker?

Answer 4

It disrupts the target gene, and enables selection of the mutant cells

Question 5

What is the end goal of a transgenic model of human disease?

Answer 5

The host chromosome ends up with the mutated version of the gene that you are studying

Question 6

Example of a property that a selection marker has?

Answer 6

It could provide resistance to a certain toxin

Question 7

How would you select the mutant cells?

Answer 7

Use the specific property of the selection marker. E.g. if it provides immunity against a certain toxin, add the toxin to the group of cells that you have

Question 8

What is done with the successfully identified mutant cells?

Answer 8

They are inserted into another blastocyst (not the one they came from) which is then transferred to a surrogate mother to develop

Question 9

How can a particular gene be deleted only in a specific organ/tissue?

Answer 9

Cre-LoxP based methodology

Question 10

Characteristic of loxP site?

Answer 10

They have two inverted repeats on either end, and a spacer sequence in the middle

Question 11

Kind of example of loxP site?

Answer 11

ATAAGCTAAATA

Question 12

What is the spacer sequence in ATAAGCTAAATA?

Answer 12

GCTA

Question 13

What are the two inverted repeats in ATAAGCTAAATA?

Answer 13

ATAA and AATA

Question 14

What happens when Cre-recombinase recognises two loxP sites?

Answer 14

It induces recombination between them, and the intervening sequence is deleted.

Question 15

What would happen to a gene of interest if it was between two loxP sites, and Cre-recombinase was added?

Answer 15

Recombination between the loxP sites would be induced, and the gene would be deleted

Question 16

What is a floxed allele

Answer 16

A gene that has had two loxP sited placed on either side of it

Question 17

How is the gene of interest only deleted in a specific tissue/organ?

Answer 17

The Cre-recombinase promoter will only be activated in a specific tissue/organ (e.g. a heart specific promoter)

Question 18

What would happen to a gene if the Cre-recombinase promoter was heart specific?

Answer 18

The Cre-recombinase would only be expressed in the heart, so the gene would only be deleted in the heart

Question 19

Example of recombination into an animal cell?

Answer 19

Factor VIII is used as a haemophilia therapeutic protein, and the gene was transfected into hamster cells–> cells would then produce the factor VIII

Question 20

Possible issue with using an animal cell to make a human protein?

Answer 20

The pattern of post-translational modification in the animal cell may be different to the pattern doen in humans