Transgenic and Knockout Mice

Question 0

what is an ‘IN VITRO’ study ?

Answer 0

-literally mean ‘within the glass’

performed not in a living organism but in a controlled environment such as a test tube or petri dish

Question 1

what is IN VIVO research ?

Answer 1

-literally mean ‘within the living’
experimentation using a whole organism that is living

it is better suited for observing the overall effects of an experiment on a living subject

Question 2

what are 2 forms of in vivo studies ?

Answer 2

animal experimentations and clinical trials

Question 3

why are mice the most commonly used mammalian species used for animal experimentation ?

Answer 3

they replaced guinea pigs because…

• smaller
• cheaper
• faster breeding

Question 4

how many more nucleotides does the human genome have compared to the mouse genome ?

Answer 4

400 million - we are much bigger and more complex

Question 5

when did humans and mice genetically diverge ?

Answer 5

about 75 million years ago

- we had a common ancestor

Question 6

how much of the human genome is similar to the mouse genome and what does it mean ?

Answer 6

99% of human genes have a mouse counterpart

only 300 genes unique to either organism

Question 7

what does “KNOCK-OUT” mean ?

Answer 7

it means a gene can be deleted in mice to determine its function- one of the simplest ways to do this

Question 8

why is the mouse a good genetic model ?

Answer 8

STRAIN- can inbreed mice which is useful because the mice are identical and therefore you minimise other effects when looking at a specific gene
GENOME- similar counterparts to humans
LIFE CYCLE- only have a 20 day gestation period and produce 4-8pups but sometimes as many as 15. also onnly have a life span of about 2 years so aging disease can be studied and females are sexually mature by 7-8 weeks
ASSISTED REPRODUCTION- ICIS- select sperm and inject directly into an egg
GENETIC MANIPULATION- interfere with genome but knockout genes, putting in mutations etc
RESOURCES- lots of info on them such as genome is fully sequenced

Question 9

what was piebaldism caused by and what does it do ?

Answer 9

caused by a mutation of the KIT gene
lack of pigementation on the forehead and stomach
present in both humans and mice

Question 10

what happens with a congenital leptin deficiency ?

Answer 10

mutation in the leptin gene causes obesity
it is a recessive mutation so to have the disease you need 2 recessive alleles
it causes clinically obesity because leptin is an appetite suppressing hormone so therefore it causes them to eat more because they lack this hormone
the same phenotype was shown in mice

Question 11

what are some similarities and differences between mouse and human brain ?

Answer 11

mouse brain is smaller than the human brain
everything in the human brain is also present in the mouse
humans have a smaller olfactory bulb because it is not as important to humans
human cortex is folded unlike the mouse and this is because it increases the surface area which is necessary because the human brain cant be too big because then childbirth would be impossible

Question 12

what does the term transgenic mice mean ?

Answer 12

refers to mice that are genetically manipulated by technology which allows integration and expression of exogenous DNA fragments into their entire genome
- DNA that wouldnt naturally be present and it is passed onto subsequent generations

Question 13

what do transgenic mice allow?

Answer 13

enable us to study gene function or regulation and to model human diseases
may confer a gain of function (may be due to overexpression) or loss of function (integrated DNA interrupts another gene )

Question 14

what manipulations of a mouse can be done ?

Answer 14

transgenics= gene over expression
knockouts= deletion of a gene
knock ins= modifications of a gene - not the same as a new gene being added, it is the addition of a mutation to an existing gene
inducibles= induce expression or knock out a gene using a drug

Question 15

how to genetically modify a mouse?

Answer 15

random integration
homologous recombination
in combination

Question 16

what does random integration entail ?

Answer 16

injection of DNA into a fertilised mouse egg so the DNA randomly integrates into the genome

• over expressiion
• region specific promoters= splice gene with specific promoter so gene is only expressed in some cell types
• inducible system

Question 17

what does homologous recombination entail ?

Answer 17

use of embryonic stem cells- target a particular sequence to knockout gene or knock in mutation
c
-consitiutive knockout
-knockins

Question 18

what does in combination entail ?

Answer 18

make a mouse express the enzyme cre by random integration and then using homologous recombination you produce a mouse with lox P sites which surround the gene of interest that we want to knock out and then these 2 mice are crossed together
you get mice which express cre in particular cell types
cre combines with lox P sites and knocks gene out

Question 19

explain the process of producing a transgenic mouse using random integration ?

Answer 19

1- transgene spliced into a plasmid
2- transgene construct is injected into the pronucleus of a fertilised egg
3- eggs are implanted into a pseudo-pregnant mouse
4- offspring are screened for the presence of the transgene
DNA randomly integrates into the genome so you produce founders that have the DNA integrated in different area

Question 20

what can transgenes carry ?

Answer 20

gene of interest
reporter construct such as GFP which can be spliced with the gene of interest so you can determine where the gene of interest is expressed
control element- cre recombinase

Question 21

what founders are crossed?

Answer 21

the heterozygous ones to try and get a homozygous

Question 22

what is a pseudo-pregnant mouse ?

Answer 22

female mated with a vasectomised male so her physiology makes her act like she is pregnant so her uterus is good to carry offspring

Question 23

how is a transgenic construct designed?

Answer 23

regulatory promoter sequence- controls when and where the gene will be expressed
cDNA of the gene- no introns, all the exons are joined together

Question 24

what is used if you want over expression of a gene?

Answer 24

unregulated strong promoter is used such as cytomegalovirus (CMV)
it is placed upstream of the injected gene

Question 25

what is used to regulate the expression of a gene ?

Answer 25

a cell specific promoter

Question 26

what is a promoter used as a cell specific promoter ?

Answer 26

glial fibrillary acidic protein (GFAP)
expressed in a cell specific manner
expressed in mature astroglial cells

Question 27

what happens to the mice produced by pronuclear microinjection ?

Answer 27

tail biopsies used to done so DNA could be isolated however now ear punches are done to isolate DNA for analyses by southern blot or PCR to identify founder mice
only 20-30% of the offspring will carry the transgene and are founders

Question 28

what does each founder have ?

Answer 28

varying number of copies of the transgene integrated at different locations of the genome

Question 29

what is done to establish lines of the transgenic mice ?

Answer 29

founder mice are mated with wild type mice - 50% of offspring will inherit the transgene

if transgenic cells contribute to the germ lie then some transgenic eggs or sperm will be produced and the next generation of mice will be fully transgenic

Question 30

what are the limitations of transgenic mice for overexpression ?

Answer 30

major limitation is the randomness of the site of integration of injected DNA
- cant be predicted or controlled

ectopic expression

• even if using an endogenous promoter the gene may not be expressed in normal places or cell populations
• may be studying artifact because the insertion of a gene can knockout another gene and this could cause a phenotype which is classed as being an effect of the insertion of this gene when in fact it is not
• leads to wrong conclusions

Question 31

what other issues and resolutions are caused by transgenic mice for over expression ?

Answer 31

copy number
- sometimes multiple copies of transgene will insert together
- different founder lines with different levels of expression may allow gene dosage studies
insertion into other genes
- random integration may disrupt function of genes in an area surrounding transgene
- characterize effect in more than one founder line

Question 32

what is a knockout mouse?

Answer 32

a genetically engineered mouse in which one or more genes have been turned off through a targeted mutation

Question 33

what are constituitive knockout mice ?

Answer 33

a neomycin resistance gene is put in place instead of the exon and therefore an exon is knocked out
DTA is put downstream of the neomycin gene to ensure homologous recombination occurs - it shouldnt be seen if homologous recombination has taken place
the homologous recombination occurs in embryonic stem cells and this is then injected into mouse blastocysts
these are implanted into a pseudopregnant female
back cross chimeric offspring

Question 34

what happens in constitutive knock in mice?

Answer 34

instead of knocking out the exon with neomycin resistance gene, the gene is still inserted but the exon is mutated
homologous recombination in stem cells and injected into mouse blastocysts
implant into pseudopregnany female
backcross chimeric offspring

Question 35

what are the main steps of gene targetting ?

Answer 35

1) generate target vector- plasmid is cut with restriction enzymes to linearise it
2) electroporate the vector into embryonic stem cells
3) culture the stem cells in the presence of a selectant and isolate the resulting colonies - only cells that contain neomycin resistance gene will grow
4) identify stem cells with desired arrangments
5) inject embryonic stem cells into blastocysts
6) identify chimeric pups
7) generate progeny that carry the targetted gene

Question 36

what are embryonic stem cells ?

Answer 36

undifferentiated pluripotent cells from mouse blastocysts which can develop ino any type of cells and organ in the body

Question 37

how are targetting vectors constructed ?

Answer 37

each end of the targeting vector should contain sequences that are the same as the chromosomal DNA surrounding the part of the locus you want to modify
there is a unique restriction enzyme site between the vector backbone and homology arm

Question 38

what do the homologous arms define?

Answer 38

the specific region of genomic integration

Question 39

what does the process of homologous recombination do /?

Answer 39

it swaps the targetting vector for the endogenous gene

- this process is rare and requires many cells to be electroporated

Question 40

how are knock in targetting vectors made?|

Answer 40

a subtle mutation is made in one of the homologous arms- point mutation, microdeletion or insertion
when the target gene is exchanged with the homologous sequence from targetting vector the mutation is introduced to the chromosome

Question 41

what will not be in the genome if recombination has occured ?

Answer 41

tk gene therefore the cells will be insensitive to gancyclovir
but if vector has been inserted completely into the genome then the cell will die when grown in gancyclovir

surviving clones are analysed for the rare event that homologous recobination has occured using pcr or southern blot

Question 42

what happens during blastocyst injection ?

Answer 42

stem cells are injected into an early mouse embryo and this is implanted into a pseudopregnant female
embryo consists of 2 types of cells- native cells of blastocyst and the injected embryonic stem cells
mouse that develops from these cells is a chimera
chimeric mice have fur of both colours and they are mated with normal mice to obtain mice that are derived exclusively from the modifie stem cells

Question 43

what strain of mousse do embryonic stem cells originate from ?

Answer 43

strain 129 with an agouti coat

blastocysts come from strain with black coat

Question 44

why are the chimeras mated with the black wild type mice ?

Answer 44

to investigate if the genetic modification can be inherited - transmitted through germ line
- offspring of this can have either black or brown coat

Question 45

what are the main steps of gene targetting ?

Answer 45

embryonic stem cels are cultured
homologous recombination is determined by selection with neomycin and ganciclovir
the knockout embryonic stem cells are injected into host blastocyst
implantation of chimeric blastocyst into foster mother
interbreeding

Question 46

what are the issues with knockouts/knock ins ?

Answer 46

embryonic lethality- no pups get born
compensatory changes
developmental/environmental effects
neomycin cassette alters expression levels

Question 47

what are the resolutions for constitutiev knockout/knockins ?

Answer 47

inducible knockout- adult onset
regional specificity- cell type specific
transgenic expression

removal of neo cassette in embryonic stem cell