EXAM 3 - Mouse Model

Question 1

Explain why we need mouse models.

Answer 1

• We cannot use humans for genetic experiments for ethical reasons
• Mice naturally develop conditions that mimic human diseases
• Mice have many advantages over other organism models

Question 2

Describe the advantages of mice over other organisms.

Answer 2

• their genome is similar to the human genome
• low cost of maintenance
• nine week generation time
• good genetic/molecular toolbox is available
• high throughput studies make it cost efficient

Question 3

Describe the situations where transgenic mice are used.

Answer 3

Characterize the ability of a promoter to direct tissue-specific gene expression
* e.g. a promoter can be attached to a reporter gene such as LacZ or GFP

Examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

Study gene function
* many human diseases can be modeled by introducing the same or similar mutations into the mouse

Do drug testing
* used for preclinical studies

Question 4

Explain how to improve reliability of mouse models.

Answer 4

Mouse models do not aim to fully model a disease or disease mechanism –> rather set out to obtain specific functional info.
* optimal genetically modified mice need to be validated, reproducible, robust, and cost-effective

Question 5

Explain the Jackson Lab.

Answer 5

Lab that produces optimal mouse models
* over 2,500 targeted and 1,200 transgenic mutatnt mouse lines representing mouse models for much of the human disease spectrum.

Question 6

List some mouse models developed and/or distributed by the Jackson Lab.

Answer 6

• Down syndrome - Ts65Dn
• Cystic fibrosis - Cftr knockout
• Cancer - p53 knockout
• Type 1 diabetes - NOD
• Type 2 diabetes - Cpefat, Lepob, Leprdb, tub
• epilepsy - swe
• heart disease - Apoe and C57BL/6L
• muscular dystrophy - Dmdmdx
• ovarian tumors - SWR and SWXJ

Question 7

Explain inbred strains of mice.

Answer 7

Mice that can be traced to a single ancestral pair and have been mated, brother to sister, for 20 generations or more.
* all inbred mice are “strains”
* outbred mice are “stocks”

Question 8

Explain the naming of newly created inbred strains of mice.

Answer 8

Uppercase letters and numbers, always beginning with a letter (e.g. CBA, DBA)
* first substrain designation is seperated from the main strain with a ”/” (e.g. C57BL/6 and C57BL/10 are both substrains of the C57BL strain)

Question 9

Explain the naming of progeny mice of two inbred stains.

Answer 9

Progeny of two inbred stains –> crossed in the same direction –> are genetically identical.

• Designated using uppercase abbreviations of the two parents (maternal listed first, followed by F1)
  D2B6F1 - Mouse that is the offspring of a DBA/2N mother and C57BL/6J father. A full F1 designation is (DBA/2N x C57BL/6J)F1
• B6D2F1 - Mouse that is the offspring of the reciprocal cross. A full F1designation is (C57BL/6J x DBA/2N)F1.
• When two F1 mice are bred, the offspring are termed F2 D2B6F2 - offspring of a B2B6F1 intercross

Question 10

Define a transgenic mouse.

Answer 10

Generic term for an engineered mouse that has a normal DNA sequence for a gene replaced by an engineered sequence or a sequence from another organism.

Question 11

Define knockout mouse.

Answer 11

A transgenic mouse in which the normal gene is missing or engineered so that is not transcribed or translated.
* “Knocks out” that gene.

Question 12

Define knockin mouse.

Answer 12

A transgenic mouse in which the engineered “transgene” is subtly manipulated to:
* (A) alter the function of the gene (e.g., replace one amino acid with another in a site to determine if that site is essential for the protein’s function)
* (B) change transcription rate to over- or under-produce the gene product
* (C) create a fluorescent gene product to map its distribution in tissue
* (D) bring in the foreign (e.g. human) gene.

Question 13

Define conditional knockout mouse.

Answer 13

A transgenic mouse in which the transgene is knocked out in specific tissues, at a specific developmental stage, or in response to an exogenous substance (e.g., an antibiotic)

Question 14

Explain the process of generating transgenic mice.

Answer 14

• Genes responsible for particular traits or disease susceptibility are chosen and extracted
• The genes are delivered into either fertilized eggs or embryonic stem cells
• Embryos are developed and implanted in the urterus of the surrogate mother.
• The selected genes will be expressed by some offspring.

Question 15

Describe the two methods of introducing foreign DNA to generate transgenic mice .

Answer 15

1. transforming embryonic stem cells (ES cells) growing in tissue culture with the desired DNA
2. injecting the desired gene into the pronucleus of a fertilized mouse egg.

Question 16

Explain the process of Method 1: transforming ES cells growing in tissues culture with the desired DNA.

Answer 16

1. fertilize the egg
2. harvest ES cells from inner cell mass of blastocyst cells.
3. grow ES cells in culture
4. select cells expressing desired gene
5. inject transformed ES cells back into the ICM of blastocysts
6. implant into uterus of surrogate
7. test offspring for presence of gene
8. mate heterozygous offspring to produce homozygous transgenic for inserted gene

Question 17

Explain process of method 2: pronuclear injection to produce transgenic mice.

Answer 17

1. inject DNA into nucleus of embryonic cell
2. re-implant modified embryo cell into pseudopregnant female mouse
3. identify transgenic progeny
4. analyze phenotype

Question 18

What are the two outcomes of a replacement gene?

Answer 18

• restores function in a mutant animal
• knocks out the function of a particular locus

Question 19

Explain the difference between random insertion and targeted gene insertion.

Answer 19

Random insertion - transgene can be integrated anywhere in the genome.
Targeted insertion - insert transgene into one specific location.

Question 20

Explain the requirements of targeted gene insertion.

Answer 20

• the desired gene
• neor, a gene that encodes an enzyme that inactivates the antibiotic neomycin and its relatives, like the drug G418, lethal to mammalian cells.
• tk, a gene that encodes thymidine kinase, an enzyme that phosphorylates the nucleoside analog gancyclovir

DNA polymerase fails to discriminate against the resutling nucleotide and inserts this nonfunctional nucleotide into replicating DNA. Ganciclovir kills cells that contain the tk cell

Question 21

Explain positive selection.

Answer 21

Positive selection involves targeting the desired cell population with an antibody specific to a cell surface marker (CD4, CD8, etc.). The targeted cells are then retained for downstream analysis.
* One of the additional genes (neoR) confers neomycin resistance.
* permits positive selection of cells in which either homologous (specific) or non-homologous (random) recombination has occured.

Question 22

Explain negative selection.

Answer 22

Negative selection is when several cell types are removed, leaving the cell type of interest untouched.
* the second gene, thymidine kinase gene from Herpes SImplex VIrus (tkHSV) confers sensitivity to gancyclovir (a cytotoxic nucloeotide analog).
* Permits negative selection of ES cells in which non-homologous recombination has occured.

(only cells that undergo homologous recombination can survive this selection scheme)

Question 23

Describe chimeras.

Answer 23

The resultant mice after ES cells containing the mutation are introduced into early mouse embryos.
* resultant mice contain tissues derived from both the transplanted ES cells and host cells.
* can contribute to germ cell and somatic cell population
* chimeric mice are mated to see whether the mutation is incorporated into the germline
* heterozygous chimeric mice produce homozygous (knockout) transgenic mice

Question 24

Explain these examples of knock out mice.

Answer 24

Question 25

Explain conditional knockouts.

Answer 25

A technique that eliminates gene expression in a specific organ, tissue, or cell at a specific time.
* whereas in traditional knockout, the gene is deleted altogether –> sometimes we don’t want to delete genes that are essential for embryonic development
* there are now techniques with which transgenic mice can be made where a particular gene gets knocked out in only one type of cell

Question 26

Describe Cre-lox P recombination.

Answer 26

Developed to delete a specific portion of DNA in P1 bacteriophages.
* in P1 viruses, there is a cre enzyme and lox P DNA sequence
* Lox P sites work in pairs and they flank the target DNA –> target DNA is deleted
* the cre enzyme cuts the lox sites in half and splices the two halves together to form one lox site.

Question 27

Explain wht occurs when CRE recombinase is expressed.

Answer 27

The gene between lox P sites is removed.

Question 28

Describe the examples of research where knockoutmuce have been useful.

Answer 28

• Obesity
• Heart disease
• Diabetes
• Arthritis
• Substance abuse
• Anxiety
• Aging
• Parkinson Disease

Question 29

Explain the drawbacks of knockout mice.

Answer 29

• Many are developmentally lethal
• Failure to produce a change
• Different change than that predicted in humans

Question 30

Describe genome engineering using the CRISPR-Cas9 system.

Answer 30

• No additional foreign DNA is introduced to the targeted gene region* Simpler and more efficient method of gene editing than used earlier
• Uniquely advantageous for modifying multiple loci at once
• Generate heritable transgenics in a single generation
• Minimize off-target gene disruptions or integration of transgenes
• Generate knock-in or knock-out cell lines or animal strains -
  Transgenic zebrafish, mice and monkeys have been developed
• Named one of the top scientific breakthroughs of 2013