L27 Genetically Modified Animals

Question 1

Why are genetically modified animals used in research?

Answer 1

to study gene function in complex organisms

Question 2

What is ‘pharming’?

Answer 2

the use of animals to produce pharmaceuticals

Question 3

What species is the most common model animal?

Answer 3

Mouse

Question 4

Why are mice good model organisms?

Answer 4

Their genome is 80-90% similar to humans and they reproduce quickly

Question 5

What is a transgenic mouse?

Answer 5

a mouse with a gene inserted randomly

Question 6

What is a knockout mouse?

Answer 6

a mouse with a gene that is specifically inactivated

Question 7

What is a knock-in mouse?

Answer 7

a mouse with DNA inserted at a specific site to modify function

Question 8

From where are embryonic stem cells harvested?

Answer 8

the inner cell mass of blastocysts.

Question 9

What stage embryo are blastocysts?

Answer 9

30-130 cells

Question 10

What characteristic makes embryonic stem cells useful for gene editing?

Answer 10

they are pluripotent

Question 11

What does the neomycin resistance gene in the plasmid do/

Answer 11

makes cells resistant to neomycin or G418

Question 12

What does the tk gene from herpes simplex virus do?

Answer 12

converts ganciclovir into a toxic compound

Question 13

How is the plasmid introduced into stem cells?

Answer 13

electroporation

Question 14

What type of recombination is used in gene targeting?

Answer 14

homologous recombination

Question 15

What does positive/ negative selection screen for?

Answer 15

correct homologous recombination

Question 16

what does neomycin sensitivity indicate?

Answer 16

no recombination

Question 17

What does ganciclovir sensitivity indicate?

Answer 17

random insertion including the tk gene

Question 18

What combination of drug resistance indicates correct recombination?

Answer 18

resistance to both neomycin and ganciclovir

Question 19

How is gene insertion confirmed?

Answer 19

PCR

Question 20

What does a single large PCR band suggest?

Answer 20

homozygous mutant

Question 21

What does two PCR bands (one normal, one larger) suggest?

Answer 21

heterozygous mutant

Question 22

Where are edited stem cells injected?

Answer 22

into another blastocyst

Question 23

What type of mice result from injection of blastocysts?

Answer 23

chimeric mice

Question 24

How is germline transmission confirmed?

Answer 24

breeding chimeric mice and genotyping offspring

Question 25

How long does the traditional knockout mouse method take?

Answer 25

8-10 months

Question 26

What is the typical cost for traditional gene targeting in mice?

Answer 26

~$10,000

Question 27

What is the function of Cas9 in CRISPR?

Answer 27

it cleaves DNA at a target sequence

Question 28

What component guides Cas9 to the correct sequence?

Answer 28

guide RNA

Question 29

What is used as a repair template in CRISPR editing?

Answer 29

an oligonucleotide or plasmid with homology arms

Question 30

At what developmental stage is CRISPR injected?

Answer 30

zygote

Question 31

Why is CRISPR more likely to result in homozygous edits?

Answer 31

Cas9 targets both chromosomes simultaneously

Question 32

How long does CRISPR editing typically take?

Answer 32

about 3 months

Question 33

What is the typical cost of CRISPR editing in mice?

Answer 33

around $5,000

Question 34

What is a mosaicism in CRISPR gene editing?

Answer 34

mixed genetic profiles due to editing after first cell division

Question 35

What is a major risk of CRISPR?

Answer 35

off-target effects

Question 36

How is gene editing introduced in adult mice?

Answer 36

using a virus carrying guide RNA

Question 37

What enzyme is used in Cre-Lox recombinantion?

Answer 37

Cre recombinase

Question 38

What are LoxP sites?

Answer 38

specific DNA sequences recognised by Cre recombinase

Question 39

What feature gives LoxP sites directionality?

Answer 39

an asymmetric central sequence

Question 40

What happens when LoxP sites re head to head?

Answer 40

DNA between them is inverted

Question 41

What happens when LoxP sites are head to tail?

Answer 41

DNA between them is excised

Question 42

What happens when LoxP sites are on different chromosomes?

Answer 42

DNA is swapped

Question 43

What is a "floxed" gene?

Answer 43

a gene flanked by LoxP sites

Question 44

What is required to knock out a gene using Cre-lox in a specific tissue?

Answer 44

a tissue-specific Cre-expressing mouse and a floxed gene

Question 45

What promoter is used to drive liver-specific Cre expression?

Answer 45

Alb promoter

Question 46

What does crossing floxed mice with Alb-Cre mice produce?

Answer 46

gene knockout in liver cells only

Question 47

What antibiotic is used for positive selection in stem cells?

Answer 47

Neomycin or G418

Question 48

What drug is used for negative selection in stem cells?

Answer 48

ganciclovir

Question 49

What gene confers ganciclovir sensitivity?

Answer 49

tk gene from herpes simplex virus

Question 50

Why is homologous recombination rare?

Answer 50

it depends on random DNA breaks aligning with homology arms

Question 51

What is the genotype of a chimeric mouse after injection?

Answer 51

a mix of modified and unmodified cells

Question 52

How are founder mice identified?

Answer 52

by PCR of offspring from breeding

Question 53

What genotype is needed for a homozygous knockout?

Answer 53

x/x

Question 54

What is the purpose of breeding heterozygotes?

Answer 54

to produce homozygous mutants

Question 55

Which method is faster: traditional gene targeting or CRISPR?

Answer 55

CRISPR

Question 56

Which editing method has a higher risk of off-target effects?

Answer 56

CRISPR

Question 57

What kind of gene can be tested using Cre under a liver promoter?

Answer 57

a gene affecting liver function

Question 58

What technique allows cell-type specific knockouts?

Answer 58

Cre-lox recombination

Question 59

What is the purpose of using genetically modified animals in pharming?

Answer 59

to produce pharmaceutical proteins in live animals