Mouse Genetics
Mouse characteristics
Powerful models of human disease – cancer, obesity, embryonic development,
behaviour
Large litter size for mammals (8)
High re-mating frequency for males (females = 3 weeks)
Easy to raise, durable
Generation time: 2-3 months
Store frozen embyros
Genome sequence completed February 2001 and published in 2002 – 98% of genes
have human homologues
BUT…
Internal fertilisation and embryos develop in utero
Too expensive time consuming for a F2 screens (limit to how many offspring you can
produce at any one time – limits to litter size)
Mouse mutants
Origins: Mouse fancying societies – 1700s hobby in Japan – collected mice with
different coloured coats, Mendel also used mice at one point to study inheritance but
was banned – it was unholy for a monk to live with animals having sex: societies were
inadvertently doing genetic experiments, selective/artificial breeding uncovered
mutant phenotypes
- Tumourgenesis: transplant one tumour from one mouse to another – but problem
is that the immune system rejects the foreign tumour (in a time when genetics
was not developed)
- Use inbred strains of mice to transplant tumours
In the lab
Pioneers William Castle and Clarence Little – first focused on coat-colour genetics
Developed inbred mouse strains still used today – important as a uniform genetic
background is needed in order to be used to compare new variations, several
hundred inbred strains
- Little’s first inbred strain DBA (dilute brown non-agouti) developed in 1909,
most famous strain C57BL/6 in 1921 (alcoholic – genome sequenced and published
in 2002)
- Inbreeding generated through selection for quantitative traits (eg. Pax6)
, Researchers focused on finding mutants and variants, then mapping the genes
involved (eg. coat colour, ear shape, tail length), emergence of biochemical markers
in the 1970s allowed protein variants to be identified
- Over 1200 mutations/variants catalogued by 1970s
- Mutants obtained through: mouse fancy societies, large scale inbreeding
programmes, mutagen risk assessment (mouse could be used as a model to study
the effects of UV radiation)
- F1 screens for dominant mutations, directed screens using deletions,
recombinant inbred strains, knockouts and transgenics (due to inefficiency of F2
screens)
Dominance and Haploinsufficiency
Haploinsufficiency: dominant phenotype in 2n organisms that are heterozygous for a
loss of function allele – though mechanisms of this are not well understood
- T/+ short tail, T/T no tail – mesodermal defects
- Sey/+, small eyes
- Sey/Sey – no eye, no nose brain defects
- In human mutatations of Pax6 (Sey) are associated with an autosomal dominant
disease (Aniridia)
Dominant mutations tend to be uncommon, but in terms of these phenotypes, the
effect of having only one functional copy of the gene is subtle – screens that focus
on looking for subtle effects…
New large F1 screens for dominant effects exploit behavioural assays or medical
technology
Behavioural assays (originally used to test
the side effects of drugs), ultrasounds,
ophthalmoscopes
Use F1 screens and different inbred
strains for efficiency
- Mutagenise males from different inbred strain and cross with another female
inbred strain
- no recombination – relatively uniform – F1 offspring are still relatively uniform in
genetic make-up (as they are homozygotes – relatively uniform – can pick up
subtle haploinsufficiency)
- Run through ultrasound and behavioural assays etc.
- Each of the new mutants is just one mouse – the mutation phenotype is not
obvious
- Identify potential mutants + confirm mutation is genetic – backcross F1
individual with parent strains – if dominant – see the mutation in 50% of
offspring
, - Backcross can also be first step for mapping the gene
40 000 F1 mice generated for screening for behavioural and other defects, confirm
genetic origin of defect by backcrossing to C3H – map using polymorphisms between
parental strains
- Screening for eye defects in the retina by opthalmoscope: 6000 mice screened,
25 mutants found
- Genes affected: collage Col4a1, growth factor receptor Egfr and homeobox gene
Phox2b
Other type of F1 screen – radiation induced mutations often involve deletions
Deletions can be mapped by cytogenetics (mouse chromosomes all look different) or
by loss of strain specific DNA markers
- Each mouse chromosome are different, distinct banding patterns, apocentric (no
cross-shaped) – a 3Mb visible deletion covers a region of Chr 4 contianing 52
protein coding genes, 39 with human homologu es
- 10-15% of mouse genome covered by deletions
- Map by looking at absence of markers
- Just having protein sequence for gene – need to have a mutation for a gene is
more useful to uncover how it works
F1 screen for directed recessive mutations
To uncover many mutations in these deletion regions
(identified above) is to set up a cross
Mutagenesis using ENU to achieve point mutations (more
precise) from male wild-type stock crossed to hemizygous
female for a deletion
- F1 - if you have by chance generated a mutation in the deleted region – then the
offspring will be hemizygous as well for the whole region but will also carry a
single a point mutation on the other chromosome – so recessive phenotype of the
locus is then revealed (as both chromosomes do not have the gene cannot
produce the protein product
- For a different mutation, use a different deletion
- Occasionally, can get dominant effects that occur outside of the deletion
backcross to show that the mutant is indeed genetic (back cross to wild-type
allows dominant and recessive effects in the F1 to be distinguished)
Newer techniques…
, Inbred strains vary for many quantitative traits
(between strains)
- Difficult to look at multifactorial or complex
traits as the genetic architecture is quite
different, too homogenised to model human
populations
To solve this…
Recombinant inbred strains – can re-create a wild-
type genetic make-up
- Recombinant inbred strains – crossing inbred strains with each other – now have
heterozygotes but we know what strains they were generated from
- Still have genetically identical populations but more recombinants – without
needing to sequence
- From each recombinant, clones can be made, putting genes in different contexts
can reveal other effects
Transgenic and knockout mice
- Transgenic mice: mouse eggs could be injected with DNA (eg. a reporter gene
construct) which was incorporated into the genome – the protein made by the
reporter gene is therefore produced in the same place, same stage of
development as the target gene of interest
- Knockout mice: Matt Kaufman, Martin Evans, Gail martin grew cells from an early
embryo, was later shown that the cells could contribute to a new embryo, grow
into an adult mouse and be part of the germline – if a gene was modified in the
embryonic stem cells, modification could be passed onto progeny generations
First knockout mice lacked the HRPT gene (hypoxanthine guanine phosphoribosyl
transferase gene) which in humans caused a mental retardation disorder Lesch-
Nyhan syndrome
These mice were produced by identifying a random mutation, but homologous
recombination technique (gene targeting) made this process more efficient
- Around 3500-4000 genes knocked out – more possibilities where gene is only
removed in certain cell types, gene turned on/off when drug added, knock-in –
replace existing gene with another gene
Genetics of Obesity and Weight Regulation
Early onset obesity and yellow coat in transgenic mice with ubiquitous expression of
a protein normally restricted to epidermis
Mouse characteristics
Powerful models of human disease – cancer, obesity, embryonic development,
behaviour
Large litter size for mammals (8)
High re-mating frequency for males (females = 3 weeks)
Easy to raise, durable
Generation time: 2-3 months
Store frozen embyros
Genome sequence completed February 2001 and published in 2002 – 98% of genes
have human homologues
BUT…
Internal fertilisation and embryos develop in utero
Too expensive time consuming for a F2 screens (limit to how many offspring you can
produce at any one time – limits to litter size)
Mouse mutants
Origins: Mouse fancying societies – 1700s hobby in Japan – collected mice with
different coloured coats, Mendel also used mice at one point to study inheritance but
was banned – it was unholy for a monk to live with animals having sex: societies were
inadvertently doing genetic experiments, selective/artificial breeding uncovered
mutant phenotypes
- Tumourgenesis: transplant one tumour from one mouse to another – but problem
is that the immune system rejects the foreign tumour (in a time when genetics
was not developed)
- Use inbred strains of mice to transplant tumours
In the lab
Pioneers William Castle and Clarence Little – first focused on coat-colour genetics
Developed inbred mouse strains still used today – important as a uniform genetic
background is needed in order to be used to compare new variations, several
hundred inbred strains
- Little’s first inbred strain DBA (dilute brown non-agouti) developed in 1909,
most famous strain C57BL/6 in 1921 (alcoholic – genome sequenced and published
in 2002)
- Inbreeding generated through selection for quantitative traits (eg. Pax6)
, Researchers focused on finding mutants and variants, then mapping the genes
involved (eg. coat colour, ear shape, tail length), emergence of biochemical markers
in the 1970s allowed protein variants to be identified
- Over 1200 mutations/variants catalogued by 1970s
- Mutants obtained through: mouse fancy societies, large scale inbreeding
programmes, mutagen risk assessment (mouse could be used as a model to study
the effects of UV radiation)
- F1 screens for dominant mutations, directed screens using deletions,
recombinant inbred strains, knockouts and transgenics (due to inefficiency of F2
screens)
Dominance and Haploinsufficiency
Haploinsufficiency: dominant phenotype in 2n organisms that are heterozygous for a
loss of function allele – though mechanisms of this are not well understood
- T/+ short tail, T/T no tail – mesodermal defects
- Sey/+, small eyes
- Sey/Sey – no eye, no nose brain defects
- In human mutatations of Pax6 (Sey) are associated with an autosomal dominant
disease (Aniridia)
Dominant mutations tend to be uncommon, but in terms of these phenotypes, the
effect of having only one functional copy of the gene is subtle – screens that focus
on looking for subtle effects…
New large F1 screens for dominant effects exploit behavioural assays or medical
technology
Behavioural assays (originally used to test
the side effects of drugs), ultrasounds,
ophthalmoscopes
Use F1 screens and different inbred
strains for efficiency
- Mutagenise males from different inbred strain and cross with another female
inbred strain
- no recombination – relatively uniform – F1 offspring are still relatively uniform in
genetic make-up (as they are homozygotes – relatively uniform – can pick up
subtle haploinsufficiency)
- Run through ultrasound and behavioural assays etc.
- Each of the new mutants is just one mouse – the mutation phenotype is not
obvious
- Identify potential mutants + confirm mutation is genetic – backcross F1
individual with parent strains – if dominant – see the mutation in 50% of
offspring
, - Backcross can also be first step for mapping the gene
40 000 F1 mice generated for screening for behavioural and other defects, confirm
genetic origin of defect by backcrossing to C3H – map using polymorphisms between
parental strains
- Screening for eye defects in the retina by opthalmoscope: 6000 mice screened,
25 mutants found
- Genes affected: collage Col4a1, growth factor receptor Egfr and homeobox gene
Phox2b
Other type of F1 screen – radiation induced mutations often involve deletions
Deletions can be mapped by cytogenetics (mouse chromosomes all look different) or
by loss of strain specific DNA markers
- Each mouse chromosome are different, distinct banding patterns, apocentric (no
cross-shaped) – a 3Mb visible deletion covers a region of Chr 4 contianing 52
protein coding genes, 39 with human homologu es
- 10-15% of mouse genome covered by deletions
- Map by looking at absence of markers
- Just having protein sequence for gene – need to have a mutation for a gene is
more useful to uncover how it works
F1 screen for directed recessive mutations
To uncover many mutations in these deletion regions
(identified above) is to set up a cross
Mutagenesis using ENU to achieve point mutations (more
precise) from male wild-type stock crossed to hemizygous
female for a deletion
- F1 - if you have by chance generated a mutation in the deleted region – then the
offspring will be hemizygous as well for the whole region but will also carry a
single a point mutation on the other chromosome – so recessive phenotype of the
locus is then revealed (as both chromosomes do not have the gene cannot
produce the protein product
- For a different mutation, use a different deletion
- Occasionally, can get dominant effects that occur outside of the deletion
backcross to show that the mutant is indeed genetic (back cross to wild-type
allows dominant and recessive effects in the F1 to be distinguished)
Newer techniques…
, Inbred strains vary for many quantitative traits
(between strains)
- Difficult to look at multifactorial or complex
traits as the genetic architecture is quite
different, too homogenised to model human
populations
To solve this…
Recombinant inbred strains – can re-create a wild-
type genetic make-up
- Recombinant inbred strains – crossing inbred strains with each other – now have
heterozygotes but we know what strains they were generated from
- Still have genetically identical populations but more recombinants – without
needing to sequence
- From each recombinant, clones can be made, putting genes in different contexts
can reveal other effects
Transgenic and knockout mice
- Transgenic mice: mouse eggs could be injected with DNA (eg. a reporter gene
construct) which was incorporated into the genome – the protein made by the
reporter gene is therefore produced in the same place, same stage of
development as the target gene of interest
- Knockout mice: Matt Kaufman, Martin Evans, Gail martin grew cells from an early
embryo, was later shown that the cells could contribute to a new embryo, grow
into an adult mouse and be part of the germline – if a gene was modified in the
embryonic stem cells, modification could be passed onto progeny generations
First knockout mice lacked the HRPT gene (hypoxanthine guanine phosphoribosyl
transferase gene) which in humans caused a mental retardation disorder Lesch-
Nyhan syndrome
These mice were produced by identifying a random mutation, but homologous
recombination technique (gene targeting) made this process more efficient
- Around 3500-4000 genes knocked out – more possibilities where gene is only
removed in certain cell types, gene turned on/off when drug added, knock-in –
replace existing gene with another gene
Genetics of Obesity and Weight Regulation
Early onset obesity and yellow coat in transgenic mice with ubiquitous expression of
a protein normally restricted to epidermis
