Transgenic Organisms

Question 1

What are the two methods of targeting the transcriptome?

Answer 1

Adding expression of a gene or gene knockdown.

Question 2

What is the problem of increasing gene expression to test function?

Answer 2

The original endogenous gene/allele is still there and has an affect on phenotype.

Question 3

What is gene knockdown?

Answer 3

Using interference RNA to inhibit translation of mRNA of a particular gene.

Question 4

What is a disadvantage of use of iRNA for gene knockdown?

Answer 4

Potential off-target effects.

Question 5

What is one way of overcome off-target effects?

Answer 5

Running several parallel experiments and comparing the phenotypes.

Question 6

What are the advantages of using morpholinos?

Answer 6

Less off-target effects as interaction is more specific. oligonucluotides using morpholinos are more resistant to endogenous nucleases.

Question 7

In what animal model are morpholinos commonly used in?

Answer 7

Zebrafish

Question 8

What are the advantages of using morpholinos?

Answer 8

Quick, cheap, resistant to nucleases, less off-target effects.

Question 9

What are some disadvantages of using morpholinos?

Answer 9

Limited to mostly frog and fish embryos.
Limited lifespan (days).
need to monitor blockage of translation using specific antibodies
Off-target effects
Amount of MO delivered into the cell can vary.

Question 10

What animal is best suited for transgene experiments?

Answer 10

Mice, they are more likely to recombine the inserted gene into their genome.

Question 11

What is a limitation of using pronuclear injection/ inserting genes?

Answer 11

There is random insertion into the genome- need to run several parallel experiments.
The endogenous gene is presents- use knockout.

Question 12

What are some characteristics of using adenoviruses?

Answer 12

No integration into the genome- lost with cell division.

Can be performed in-vivo.

Question 13

What are some characteristics of using lentiviruses?

Answer 13

Transfect dividing and non-dividing cells.

Random integration into the genome.

Question 14

What percentage synteny is there between mice and humans?

Answer 14

90%

Question 15

What are the four steps of making gene knock-out mice?

Answer 15

1. In vitro mutation of the target gene in ES cells.
2. ES cells with mutation introduced into blastocyst.
3. Chimeric mice are mated to assess whether the mutation has been incorporated into the germ line.
4. Heterozygous mice are mated to make homozygous mutants.

Question 16

What is the first step to producing cells containing gene knockout?

Answer 16

1. Work out which exon to disrupt from the gene to be disrupted. Add neomycin resistance gene (green) for +ve selection.

Question 17

What is the second step to producing cells containing gene knockout?

Answer 17

Add a Thymidine kinase gene for -ve selection.

Question 18

What is the third step to producing cells containing gene knockout?

Answer 18

Isolate, maintain and grow ES stem cells outside of the mouse.

Question 19

What is the forth step to producing cells containing gene knockout?

Answer 19

Electroporate vector into ES cells.

Question 20

What are the advantages of gene insertion?

Answer 20

Can knockout the target endogenous gene and mutate the new gene.

Question 21

What are the disadvantages of using gene insertion?

Answer 21

Random insertion occurs.
15-40% of knocked out genes are lethal- difficult to study the protein at different stages.
Neomycin resistance genes can influence the phenotypes.

Question 22

What is conditional gene knockout?

Answer 22

Inserting a gene that will cause inactivation of a particular gene at a particular time/tissue. Once the conditional knockout is created, the homozygote is crossed with the recombinase.

Question 23

What is the major advantage of conditional knockout?

Answer 23

The gene is there during development and so knockout is not lethal.

Question 24

What are knock-in mice?

Answer 24

Recombination in ES cells to insert genes.