Animal models

Question 1

Draw a mouse. How many paws does it have?

Answer 1

4, if lucky

Question 2

What does it mean if the strain is inbred?

Answer 2

it is homozygous at all loci

Question 3

What does it mean if the strain is outbred?

Answer 3

goal is heterozygosity at all loci

Question 4

What does it mean if two strains are conplastic?

Answer 4

differ by the MITOCHONDRIA

Question 5

What does it mean if two strains are consomic?

Answer 5

differ by a single CHROMOSOME

Question 6

What does it mean if two strains are congenic?

Answer 6

differ by a CHROMOSOMAL STRETCH

Question 7

What does it mean if two strains are coisogenic?

Answer 7

differ by a single MUTATION

Question 8

What is a recombinant inbred strain?

Answer 8

differs from each parental strain by half the genome

Question 9

Inbred strains are generated by mating 1 pair of siblings each generation. How many generations are needed to be allowed to officially call the strain inbred?

Answer 9

20

Question 10

In outbred strains, we want to get as much diversity as possible, ideally heterozygosity for every locus. How can we achieve that?

Answer 10

a) random mating of >100 breeding pairs

b) rotation systems (>25)

Question 11

How do you create a consomic strain?

Answer 11

you want a single chromosome from a donor strain
= backcross the recipient strain with a donor strain >10 times

check with the collection of small number of markers and subsequent inbreeding

Question 12

Who is the mitochondrial donor when creating conplastic strains?

Answer 12

mother in backcrosses

Question 13

In which disease was it shown that mitochondrial DNA of the mouse strain used influences the immune response?

Answer 13

EAE - model for MS

experimental autoimmune encephalomyelitis

Question 14

What is the difference between generating consomic and congenic strains?

Answer 14

selection markers

both created by 10+ backcrosses to an inbred strain

Question 15

How can you create a coisogenic strain?

Answer 15

• introduce the mutation into the genome (by knock in/out, CRISPR…)
• separation from background strain if needed
• inbreeding

Question 16

Explain how are recombinant inbred strains created. Which project was working on that, what was their aim, approximately how many strains did they create?

Answer 16

1. breed 2 parental inbred strains (A and B) -> get AB F1 offspring
2. mate siblings (random two)
3. continue for 20 generations, until you get fully inbred recombinant strain

Project: collaborative cross
Aim: creating novel inbred strains from 8 known strains that would capture 90% of known variation
#: ca. 500 (?)

Question 17

When creating an inbred mouse, why do we need to breed it for so many generations?

Answer 17

after every generation, 12,5 % alleles get fixed (homozygous)
-> after 20 generations, you come to 98,7%

Question 18

What is the F2 analysis?

Answer 18

finding genetic differences between the offspring with a particularly interesting phenotype and the ones without by inbreeding them until being able to pinpoint a specific allele responsible for it

Question 19

Name/describe the 5 laws of transplantation (skin).

Answer 19

1. isografts (between genetically identical mice) succeed
2. allografts fail
3. transplanting skin from parent to progeny succeeds, 4. but progeny to parents does not
   - progeny is AxB = knows both
   - parents are either A or B = one of the MHCs will be recognized as foreign and the graft will be rejected
4. F2(AxB) or subsequent will more likely than not reject the graft from A (or B)
   - original F1 is most diverse
   - F2 - Fn crosses are getting more and more inbred

Question 20

What did Medawar’s, Mitchinson’s and Miller’s experiments show about specificity of tissue rejection?

Answer 20

• immunological memory (second graft of the same kind is rejected faster because memory lymphocytes have been formed against its antigens)
• grafted tissue is recognized with specific receptors
• these receptors are on lymphocytes (if you transfer lymphcytes - not serum only - you get a faster rejection of 2nd graft)
• they are distributed systemically (and are migratory)
• these lymphocytes are T cells (rejection does not happen in neonatally thymectomized animals)

Question 21

When creating congenic mouse strains for a dominant mutation, what percentage of offspring has a wanted allele fixed?
How many generations are needed to achieve 99,9% homozygosity?

Answer 21

50%
(you either have it or you don’t; and you always backcross with the original background strain)

10 generations

Question 22

How do you create a congenic mouse strain for a recessive mutation?
What is one of the issues when creating them? Which strain had a problem with this?
Who was a Nobel prize for this awarded to and what did he discover?

Answer 22

cross P0
get F1 -> cross to get F2
choose those who are homozygous for a mutation, cross with background strain again
repeat
(takes twice as long)

the closer the two genes, the lower the probability of homozygosity (because the recombination frequency between them is lower)
CD45.1 had a new point mutation that was carried on and altered susceptibility to infection with mCMV

Snell -> identified H-2 complex in mice (homologus to HLA in humans)

Question 23

What happens when:

a) skin transplant between 2 syngenic mice?
b) skin transplant between mice that have the same minor, but different major histocompatibility complex?
c) skin transplant between mice from 2 different strains, but with the same MHC alleles?
d) skin transplant if two mice are identical except for 1 or 2 minor histocompatibility complex loci?
e) skin transplant if two mice are identical except for 3/+ minor histocompatibility complex loci?

Answer 23

a) accepted
b) rejected
c) rejected as in b, because minor antigens are different
d) very slow rejection
e) rejected as in b

Question 24

Which minor antigen was shown to play a role in human transplants?

Answer 24

male antigen in kidney transplants

male to female transplants were rejected significantly faster

Question 25

What are minor histocompatibility antigens?

Answer 25

self-peptides from genes of which the recipient is not tolerant (polymorphisms or absence of Y chromosome in females)
presented on MHCI

Question 26

Why do bone marrow transplants lead to different results than skin transplants?

Answer 26

skin has no activating receptors for NK cells, so they do not play a role in skin transplants

macrophages/monocytes/T/B cells do express activating receptors and even more importantly, if the MHCI is different than the donor’s, the recipient’s NK cells do not recognize it and see it as “missing self” (no inhibitory signal from recognizing MHCI they are familiar with)

sidenote: NK CELLS ARE RADIORESISTANT

Question 27

What is inbreeding depression?

Answer 27

creating homozygous loci leads to lethal mutations

Question 28

N: How could one correct the immune-response deficiency in Eα minus C57BL/6 strain?

Answer 28

Generation of transgenic animal

1. A female mouse is injected with follicle-stimulating hormone and chorionic gonadotropin to induce superovulation and ready to mate
2. mating
3. Fertilized eggs are removed from the female, DNA containing the Eα gene is injected into the male pronucleus
4. injected eggs are transferred into the uterus of a pseudopregnant female
5. result: chimeras-
   some offspring will have incorporated the injected Eα gene (transgene)
6. mate the transgenic animal to Eα minus C57BL/6 mouse
7. Continue backcrossing for 10-20 generations to produce a strain expressing the Eα transgene
8. –> assess functions of transgenes in homogenous genetic background

Question 29

N: 1. Which characteristics do RAG-1 deficient mice show?

Answer 29

No RAG-1 means no VDJ recombination of T and B cells.

This means that in the TCR, the beta chain is not rearranged and not expressed. We don’t get a preTCR and a proliferation signal which leads to death
== No cells that would become DP, only DN T cells.

For B cells it’s similar, the heavy chain doesn’t rearrange, so we don’t even get to the preBCR (and large preB cell stage). –> stay at the early proB cell stage.