P1246341514fKOoY

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Mouse Genome Project
The mouse genome is about 14 smaller than the
human genome (2.5 Gb compared with 2.9 Gb).
approximately 98 of the genome now
available. The mouse and human genomes each seem
to contain about 30,000 protein-coding genes.
The proportion of mouse genes without any
homologue currently detectable in the human
genome (and vice versa) seems to be less than 1.
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Advantages of mouse as a model
Great degree of conservation to human genome.
The mouse is the most well developed system for
mammalian genetic analysis. Stable inbred
strains and relative short generation cycle.
Genetic data base - mouse genome sequence -
mouse ESTs - BAC library Reverse genetics
(gene targeting)
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Project Study of mouse homolog(s) (heartin) of
a human secreted factor expressed in heart
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Reverse Genetics

• The experimental procedure that begins with a
  cloned segment of DNA, or a protein sequence, and
  uses this (through directed mutagenesis) to
  introduce programmed mutations back into the
  genome in order to investigate function.
• Cloning of DNA sequence (Ge )
• Mapping of gene (Inés)
• Generation of mutant alleles (Ngozi)
• Functional analysis (study of mutant
  phenotype) (Prabhat)

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Bioinformatics approach
Mouse Genome database search using the human cDNA
sequence in search for the mouse homologs
Results If we find the mouse homologs -
Genomic sequence, map location known - Clone
the mouse homologs - Go straight to functional
study If we only find mouse ESTs in the
database then
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Clone unknown mouse cDNA Clone from mouse cDNA
library - Label probes (human cDNA sequence or
mouse EST) - Positive clones from mouse heart
cDNA library - 5 RACE RACE full length cDNA
sequence - Design primers based on human cDNA
or mouse EST sequence. - 3 and 5 RACE
full length cDNA sequence. Results Found 4
mouse homolog cDNAs
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How to choose which gene to study
Sequence analysis Result One cDNA without
signal peptide Expression pattern analysis
Northern blot to detect heart specific
expression base on gene specific probes
RT-PCR detection with gene specific
primers Results Select cDNA expressed in
the heart
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Clone mouse gene sequence
Screen mouse BAC library - Label mouse cDNA
(full length) probe - Screen positive clones
from mouse BAC library - Shotgun cloning - DNA
sequencing and assemble full gene sequence.
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Mapping of heartin gene
Why mapping ?
Already described mutations and phenotypes in
the same region
How?
Mouse Genome Database (sequence homology,
syntenic regions)
exact map position
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Mapping (contd.)
Other approaches
-Somatic cell hybrid analysis (mouse-hamster) -Fl
uorescent in situ hybridization (FISH)
Chromosomal location Allow mapping of
non-polymorphic loci
-Formal linkage analysis mapping panels
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Mapping (contd.)
Formal linkage analysis
Mapping panels set of DNA samples from animals
carrying recombinant chromosomes
300 markers already mapped and positioned in
each chromosome
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Where our gene maps? Linkage analysis
Map Manager (Software)
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Mapping (contd)
Mapping panels most commonly used
-Recombinant inbred strains -Interspecific
backcross
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Recombinant inbred strains
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Interspecific backcross
Mus musculus X Mus spretus
Evolutionary divergent species but F1 fertile
females
polymorphism
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Find a polymorphism in our locus (RFLP, SSCP, SNP)
Select strains and Map!
ACME Labs
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Production of null mutant allele

• Design targeting vector
• - flanking DNA that is identical in sequence
  to the targeted locus
• - positive selection marker e.g. neo located
  inside the region of
• sequence similarity between the vector and
  the targeted locus
• - negative selection marker e.g. thymidine
  kinase (tk) located outside
• the region of sequence similarity between
  the vector and the targeted
• locus

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Production of null mutant allele contd.
Tanaka, M. et. al., 2000
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Production of null mutant allele contd.

• Culture ES cells
• - isolate ES cells from a wild type agouti mouse
  
• - culture on mouse embryonic fibroblast feeder
  layers in the presence
• of high-glucose medium, fetal calf serum
  and leukemia inhibitory
• factor (LIF)
• Introduce targeting vector into ES cells
• - electroporate ES cells with targeting vector,
  culture, and select
• clones using G418 and gancyclovir
• Genotype ES clones
• - southern blot analysis using 5 probe

Tanaka, M. et. al., 2000
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Production of null mutant allele contd.
Griffiths, A.J.F. et. al., An Introduction to
Genetic Analysis
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Production of null mutant allele contd.
knockout mouse (25)
genotype
genotype
Griffiths, A.J.F. et. al., An Introduction to
Genetic Analysis
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Production of conditional mutant allele

• Useful for circumventing early lethal phenotypes
• Allows biological questions to be addressed with
  exquisite accuracy
• Two members of the integrase family are used at
  present to conditionally control gene expression
  by site-specific DNA recombination
• - Cre (from bacteriophage P1)
• - Flp (from Saccharomyces cerevisiae)
• Cre and Flp recombinases catalyse a conservative
  DNA recombination event between two 34-bp
  recognition sites, loxP and FRT, respectively

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Production of conditional mutant allele contd.

• Design conditional gene targeting construct

heartin locus
Targeting vector

• Electroporate ES cells
• Select with G418 and gancyclovir
• Genotype clones by Southern blot

Targeted locus

• Flp recombinase
• Genotype clones by Southern blot

Targeted locus after neo removal
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Production of conditional mutant allele contd.

• Produce mice heterozygous for the conditional
  mutant allele
• - Inject ES cells (from brown mouse) into black
  female
• mouse blastocyst
• - Transfer blastocyst to pseudopregnant black
  female
• mouse
• - Cross male chimera progeny produced to a black
  wild-
• type female mouse
• - Genotype brown mice progeny by PCR and/or
  Southern Blotting to
• determine which mice are heterozygous for
  the conditional mutant
• allele (note black mice will not have
  targeted locus because the ES
• cells came from the brown mouse)

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Production of conditional mutant allele contd.

• Design transgene construct
• Make transgenic mouse
• - Use restriction enzymes to cut out plasmid DNA
  before injection
• - Remove zygotes from the oviduct of a female
  mouse (black) mated the night
• before
• - Inject transgene into the male pronucleus in
  zygotes
• - Transfer injected zygotes to pseudopregnant
  female mouse (black)
• - Genotype F1 progeny by PCR and/or Southern
  Blotting using probes
• against CRE-ER
• - Carry out Western Blotting and use a reporter
  system (e.g. lacZ) to determine that
• CRE-ER is functional
• - Cross hemizygous progeny to get homozygous
  transgenics

cut
cut
Cross with conditional knockout line
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Detailed loss of function phenotypic analysis -
first step to find the function
Finding the function of hertin gene

• Morphological abnormalities- presence/absence,
  size, pattern etc
• - histological/cytological analysis
• Due to? - cell death/cell proliferation/cell
  migration/fate change
• Molecular markers - in situ analysis
• Physiological changes

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Further functional analysis of hertin

• Cell autonomy/ non autonomy
• Ectopic gene expression
• Pathway Partners

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1. Cell autonomy/ non autonomy
Why? - hertin is putative secreted factor
How? - two key experiments a) wt cells in mutant
background ---- ? b) mutant cells in wt
background ---- ? Distinguish between mutant
cells and wt cells is critical - marker - lacZ

• Tools in Mice?
• Chimera Analysis
• Morula aggregation
• ES cell injection

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Let us say there is no heart in the hertin mutant
embryos
hertin ES cells to wt blastocyst embryos
1. Heart with both wt mutant cells - non
autonomous
-/- ,Rosa26lacZ ES cells
2. Heart with only wt cells - autonomous
/
wt ES cells to hertin blastocyst embryos
1. Heart with all wt cells - autonomous
/, Rosa26lacZ ES cells
2. Heart with both cells - non autonomous
-/-
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2. Ectopic gene expression
Why? To test if the function of hertin is
instructive or sufficient.

• How? - Transgenic Approach
• Microinjection - Zygote injection
• Embryonic stem cells
• - use of different or tissue specific promoter to
  drive the gene to ectopic place or time.

• Results? - depending on lof phenotype
• ectopic hearts or tissues or bigger heart? or
  some cardiac effects?
• or no phenotype?

/
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3. Pathway Partner Analysis
Where does hertin function in the pathway? What
are upstream or downstream factors?

• Gene expression analysis - in situ microarray
  analysis
• Double mutant analysis
• Modifiers - similar to enhancer/suppressor screen
• Biochemical approaches

hertin
?