Genetic Engineering

Question 1

Name four types of genetically engineered mice

Answer 1

1. Knock-out
2. Transgenic
3. Conditional Knock-out
4. Knock-in

Question 2

What is the difference between a knock-out mouse and a transgenic mouse?

Answer 2

Transgenic mice means that the mice having foreign gene where as knock out mice do not have a particular gene or it has been made non functional.

Question 2

Why would scientists make these sketchy mice anyway?

Answer 2

Because it is an ethical way to study gene function and model human diseases.

Question 3

What does “Cre” stand for, and what is its function?

Answer 3

DNA recombinase (an enzyme) that catalyzes recombination between two LoxP sites

Question 4

What does Neo R stand for?

Answer 4

Neomycin-resistance gene

Question 5

What does TK stand for

Answer 5

Thymidine kinase gene (found in Herpes virus)

Question 6

Why is TK and Neo R inserted into the target gene sequence?

Answer 6

To make a targeting vector sequence

Question 7

What is a vector?

Answer 7

DNA molecule (often plasmid or virus) that is used as a vehicle to carry a particular DNA segment into a host cell as part of a cloning or recombinant DNA technique.

Question 8

What is negative selection?

Answer 8

hinders the spread of deleterious alleles

Question 9

What is Positive selection?

Answer 9

Promotes the spread of beneficial alleles

Question 9

When adding Neomycin to a dish of Neo R is it a positive or negative selection?

Answer 9

Positive, because all of the cells without Neo R die.

Question 10

How do you make a knock-out mouse?

Answer 10

1. Designing the targeting vector
2. inserting the targeting vector into the ES (embryonic stem cells) cells via electroporation
3. selecting cells (the ones that have successfully incorporated the target vector)
4. injecting cells into a new embryo
5. breading

Question 11

When adding ganciclovir (antiviral) to a dish of TK is it a positive or negative selection?

Answer 11

Negative, because the cells with TK die.

Question 12

What is CreLoxP system

Answer 12

-Deletion of a gene in selected cell types (conditional knockout)
-Labeling of certain neuronal populations (transgenic mice)
-Allows spatial AND temporal control of genetic manipulations (e.g. tamoxifen induced CreER system)

Question 13

What is floxing a gene?

Answer 13

Deleting the target gene

Question 14

What is CRISPR

Answer 14

Clustered regularly interspaced short palindromic repeats

Question 15

What is Cas 9?

Answer 15

An enzyme that cuts DNA at a specific site within a larger recognition sequence, or target site.

Question 16

How many nucleotide sequences are in the recognition site?

Answer 16

The Cas9 recognition sequence includes a 20-nucleotide sequence called the protospacer that is determined by a guide RNA (sgRNA) bound to the enzyme

Question 17

What is sgRNA (Single guide RNA)

Answer 17

An engineered form of guide RNA that forms a complex with Cas9. The sgRNA is an approximately 100 nucleotide–long

Question 18

What is the guide region?

Answer 18

A typically 20-nucleotide region that is complementary to the target DNA sequence and that defines where Cas9 cuts. Scientists can easily customize this sequence for their own targets

Question 19

What is the Scaffold region?

Answer 19

A region that forms a multi–hairpin loop structure (scaffold) that binds tightly in a crevice of the Cas9 protein. The sequence of this region is typically the same for all sgRNAs (generic)

Question 20

What are the 2 parts of the sgRNA?

Answer 20

1. Guiding region
2. Scaffold region

Question 21

What is PAM?

Answer 21

A sequence motif immediately downstream of the protospacer sequence in the Cas9 recognition sequence that is required for Cas9 function. Cas9 recognizes the PAM sequence 5’-NGG where N can be any nucleotide (A, T, C, or G). When Cas9 binds the PAM, it separates the DNA strands of the adjacent sequence to allow binding of the sgRNA. If the sgRNA is complementary to that sequence, Cas9 cuts the DNA.

Question 22

What is target DNA?

Answer 22

The sequence of DNA that will be cut by Cas9 (this is chosen by the scientist; typically the gene of interest)

Question 23

How long does it take to make a traditional
ko?

Answer 23

2-3 years

Question 24

How long does it take to make a mouse with CRISPR?

Answer 24

4-8 weeks