Transcript Roberta Rivi, MD - Harlem Children Society

Mouse mutagenesis
Roberta Rivi, MD
Laboratory of Molecular Embryology
Mutagenesis?
• Goal: determination of the function of every
gene (functional genomics)
• Gene-driven
– also called reverse genetics
• Phenotype-driven
– also called forward genetics (classical
approach)
Mutagenesis strategies
Mutagenesis
strategy (type)
Mutagenesis
frequency
Spontaneous
Primary advantages
Primary disadvantages
5 x 1 0-6 per locus Point m utations, small deletions, chromosomal
rearrangements, and insertions of endogenous
retrovirus-like sequences.
Visible phenotypes; only requirement
is observant mouse hand lers.
Only visi ble pheno types detected, at
very l ow frequency.
X-ray
13Š50 x 10-5 per
locus
Chromosomal rearrangements: ranging from
simple deletions, inversions and translocations, to
complex rearrangements.
Rearrangements act as a molecular
landmark for cloning.
Multiple genes affected, ha rd to
dissect individual gene fu nction.
Chlorambucil (c hemical
mutagen)
127 x10-5 per
locus
Chromosomal rearrangements, especially s maller
deletions (100Š500 kb) and translocations.
Same as X-ray, but h igher
mutagenesis frequency.
Multiple genes affected, ha rd to
dissect individual gene fu nction.
Ethylnitrosourea
(chemical m utagen)
150 x 10-5 per
locus
Primarily generates point m utations, occasionally
very s mall deletions (20Š50 bp).
Single-gene mutations, amenable to
high thro ughput .
No molecular landmarks for cloning.
Transgene/
retroviral (insertional
mutagen)
5Š10% of
transgenic
animals
Disrupts endogenous gene expression or coding
sequence. Sometimes causes c hromosomal
rearrangements.
Provides a molecular landmark for
cloning.
Labour-intensive, not a pplicable to
high-throughp ut approaches.
Gene targeting
(insertional muta gen)
Almost 100% of
transgenic
animals*
Generates insertions or deletions, as designed.
Can design type of mutation as
required.
Requires k nowledge of gene and its
structure , labour-intensive,
unpredictable phenotypes.
Trapping (insertional
mutagen)
Almost 100% of
transgenic
animals*
Disrupts endogenous coding sequence.
Forward-genetic strategy, easy to
clone muta ted gene, reports
endogenous gene-expression pattern.
Unpredictable phenotypes.
* Requires pre-screening of
embryonic stem cells in vitro.
Type of mutation induced
Insertional mutagens
Gene
targeting
Generates
insertions or
deletions, as
designed.
Can design type of
mutation as required.
Requires
knowledge of
gene and its
structure, laborintensive,
unpredictable
phenotypes.
Trapping
Disrupts
endogenous
coding
sequence.
Forward-genetic
strategy, easy to
clone mutated gene,
reports endogenous
gene-expression
pattern.
Unpredictable
phenotypes.
Homologous recombination
HSV-tk
Homology
arm
Homology
arm
Modified from http://cancer.ucsd.edu/tgm/genetargeting.asp
Conditional?
• Early lethality does not allow to study the role
of a gene in organogenesis
Conditional?
• Early lethality does not allow to study the role
of a gene in organogenesis
• Tissue-specificity of gene ablation is
dependent upon the expression of Cre
Conditional?
• Early lethality does not allow to study the role
of a gene in organogenesis
• Tissue-specificity of gene ablation is
dependent upon the expression of Cre
– One contruct x many Cre lines
Phage P1: Cre-loxP system
http://bioweb.wku.edu/courses/biol22000/16Recombination/Fig.html
Insertional mutagens
Gene
targeting
Generates
insertions or
deletions, as
designed.
Can design type of
mutation as required.
Requires
knowledge of
gene and its
structure,
labour-intensive,
unpredictable
phenotypes.
Trapping
Disrupts
endogenous
coding
sequence.
Forward-genetic
strategy, easy to
clone mutated gene,
reports endogenous
gene-expression
pattern.
Unpredictable
phenotypes.
Gene-trap
Gene-trap
Gene-trap
Phenotype driven mutagenesis: chemical
mutagenesis
Spontaneous
5 x 10-6
per
locus
Point mutations, small
deletions, chromosomal
rearrangements, and
insertions of endogenous
retrovirus-like sequences.
Visible
phenotypes; only
requirement is
observant mouse
handlers.
Only visible
phenotypes
detected, at very
low frequency.
X-ray
13–50
x 10-5
per
locus
Chromosomal
rearrangements: ranging
from simple deletions,
inversions and
translocations, to complex
rearrangements.
Rearrangements
act as a
molecular
landmark for
cloning.
Multiple genes
affected, hard to
dissect individual
gene function.
Chlorambucil
127
x10-5
per
locus
Chromosomal
rearrangements, especially
smaller deletions (100–500
kb) and translocations.
Same as X-ray,
but higher
mutagenesis
frequency.
Multiple genes
affected, hard to
dissect individual
gene function.
Ethyl-NitrosoUrea
150 x
10-5
per
locus
Primarily generates point
mutations, occasionally very
small deletions (20–50 bp).
Single-gene
mutations,
amenable to high
throughput.
No molecular
landmarks for
cloning.
N-ethyl-N-nitrosourea (ENU)
• We are using the chemical N-ethyl-N-nitrosourea
(ENU) to saturate wild type chromosomes with point
mutations.
• By determining the function of genes on a mouse
chromosome, we can extrapolate to predict function
on a human chromosome.
• We expect many of the new mutants to represent
models of human diseases such as birth defects,
patterning defects, growth and endocrine defects,
neurological anomalies, and blood defects.
Chemical mutagenesis
Modified from RIKEN (The Institute of Physical and Chemical Research)
http://www.gsc.riken.go.jp/Mouse/AboutUs/overview.htm
Why Use ENU as a Mutagen
• ENU is an alkylating agent that is a powerful mutagen in
mouse spermatogonial stem cells, producing single locus
mutation frequencies of 6 X 10-3 to 1.5 x 10-3, equivalent
to obtaining a mutation in a single gene of choice in
one out of every 175 to 655 gametes screened.
• Because it is a point mutagen, ENU can induce many
different types of alleles. Loss of function mutations, viable
hypomorphs of lethal complementation groups,
antimorphs, and gain-of function mutations have been
isolated in mouse mutagenesis screens.
• Missense changes are a common finding in many human
disease mutations, therefore the ENU mutations will
complement and extend the information provided by
targeted gene disruptions.
Screening for phenotypes
Mapping
Line 04/004
(e16.5)
LICIA - MATT
446.4 unaffected
44.6 affected
Smaller embryo
Severe microcephaly and micrognathia
Abnormal limb morphology & orientation
exhibits also a general defect in Endochondral Ossification (all long bones affecteD)
Line 04/004
LICIA - MATT
131.5
Alizarin Red: stains Bone
Alcian Blue: stains Cartilage
131.6
Line 04/014
LICIA - GIUSEPPINA - BETTA
unaffected
affected
mx
mb
429.1
tc
Rudimentary or hypoplastic mx (maxilla) and mb
(mandible - jaw)
Absent hyoid
Rudimentary tracheal cartilage
Alizarin Red: stains Bone
Alcian Blue: stains Cartilage
mx
430.3
mb
hy
tc
Pax3
Snail
Mermaid (merm)