Genetic
approaches
and
analysis of
gene functionstrategies
for
manipulation
of
gene expression
Peter DOVČ, UL BFLjubljana, Nov. 2010
1.
Which
approaches
are possible
in modern
genetics?2.
Review of insertional
mutagenesis
3.
Homologous recombination4.
Gene targeting
5.
Embryonic stem cells6.
Production of chimeras
7.
Production of K.O. transgenic lines8.
Development of animal models with silenced genes
9.
Applications of K.O. animals in medical and basic research10.
Basics
of
RNAi
11.
Verctors
enabelig
gene suppression
in vivo12.
Examples
of
successful
gene knock
down
13.
Prospectives
for
medicine14.
QTL concept
15.
Association
studies16.
Candidate
gene approach
17.
Transcriptomic
approach18.
Data
driven
approach
Learning cycle
Public
perception
on genetics
1.
Pomen mutacij za odkrivanje funkcije genov
2.
Možne strategije pri odkrivanju funkcije genov
3.
Karakterizacija mutacij
4.
Nove metode za študij funkcije genov
FORWARD GENETICSKoncept genetske analize, ki za izhodišče vzame fenotip (mutacijo) in nato išče ustrezen genotip z uporabo pozicijskega kloniranja ali z analizo
kandidatnih genov.
McManus & Sharp 2002
Pregled genetskih konceptov, ki jih uporabljamo za analizo funkcije genov
Mullerjeva
shema za izolacijo recesivnih letalnih mutacij na X kromosomu Drosophile
melanogaster
Primerjava splošne mutageneze, ki jo pogosto uporabljamo znotraj koncepta “Forward
genetics”
in usmerjene mutageneze, ki jo
uporabljamo v kontekstu “Reverse
genetics”.
Genetsko presejavanje
mutantnih fenotipov: pri N. crassa so z
zbiranjem mutant z aberantno rastjo izolirali gene, ki
uravnavajo rast in razvejanje hif. V tem primeru so si pomagali z mutagenezo
haploidnih celic, ki
takoj pokažejo fenotip, ki je posledica mutacije. Med identificiranimi geni so geni za aktin, dinaktin, dinein
in druge
proteine celičnega skeleta.
Za analizo mutant genov, ki uravnavajo celični cikel (cdc
mutante) sta Hartwell
in Nurse
dobila Nobelovo
nagrado. Ker so številne mutacije te vrste letalne, sta proučevala pogojne, temperaturno senzitivne mutante. Homologni
geni pri
človeku so pogosto vpleteni v razvoj raka.
Danio rerio
Weissherbst: zmanjšana količina hemoglobina v krviHagoramo: prekinjen progast vzorec
Mutageneza
samčkov, paritev z normalnimi samicami, paritev F1 samcev z normalnimi samicami in F2 intercross.
Molekularne značke
•
Retroviralni
vektorji poškodujejo gostiteljske gene in na mestu integracije pustijo “značke”, ki nam pomagajo pri amplifikaciji
okoliških
zaporedij s PCR.
Enhancer
trap
REVERSE GENETICSKoncept genetske analize, ki za izhodišče vzame genotip (nukleotidno
zaporedje) in nato analizira fenotipske
spremembe, ki jih povzročijo spremembe nukleotidnega
zaporedja. Najpogosteje uporabljene tehnike so tehnike genskega inženirstva in homologna
rekombinacija v embrionalnih matičnih celicah
McManus & Sharp 2002
FORWARD GENETICSA genetic analysis that proceeds
from phenotype to
genotype by
positional
cloning or candidate
gene
analysis.
REVERSE GENETICSA genetic analysis that proceeds
from genotype to
phenotype by
gene- manipulation techniques,
such
as homologous
recombination in
embryonic
stem cells.
McManus & Sharp 2002
First generation
of
transgenics
was
produced by
random
insertion
of
heterologous
genes
into
the
mouse
genome
1982 R. Brinster
and
R. Palmiter produced
first
transgenic
mice
having
potential
for
agricultural applications. Litter
mates
differed
in body
size
by
2.5 fold due
to the
random
integration
of
the
human GH gene under transcriptional
control
of
the
methallothionein
promoter.Brinster et al., 1982
Rationale
behind
homologous recombination
vectors
Targeted
region
Insert
Negative marker
X X
tk
Positive
marker:Neo, Luc, GFPFunctional
gene, exon
Presence
of
positive
and
negative marker
allows
double
selection
for homologous
recombination. Screning
for
presence
of
a positive
marker
is
done by
G418 or phenotypically; screening
for
absence
of
negative marker
is performed
using
gancyclovir.
•
Cultivation of ES cells enables genetic modifications, efficient selection and preserves totipotency
in vitro.
•
Microinjection of genetically modified ES cells into recipien
blastocysts
causes formation of chimeras.
Using
coat
colour
markers, chimerism can
easily
be
detected
phenotypically
•
Chimera
for
tyrosinase gene in epithelial
cells.
Brem et al., 1992
FRONTIERS IN BIOSCIENCE;
Database; Gene Knockout http://www.bioscience.org/knockout/knochome.htm
GENE KNOCKOUTS WHICH ARE COMPATIBLE WITH VIABILITYGENE KNOCKOUTS WHICH RESULT IN PRENATAL MORTALITYGENE KNOCKOUTS WHICH RESULT IN POSTNATAL MORTALITYGENE KNOCKOUTS WHICH RESULT IN PERINATAL MORTALITY
Gene knockouts classified according to the viability of the mice:
CARDIOVASCULAR SYSTEMDIGESTIVE SYSTEMENDOCRINE SYSTEMEYEGASTROINTESTINAL TRACTHEMATOPOEITIC SYSTEMIMMUNE SYSTEM AND INFLAMMATORY RESPONSESMUSCULOSKELETAL SYSTEMNERVOUS SYSTEMORGANS OF SPECIAL SENSEREPRODUCTIVE SYSTEMRESPIRATORY SYSTEMSKIN AND/OR INTEGUMENTURINARY SYSTEMMULTIPLE ORGAN SYSTEMS
STAT5ADefect in granulocyte-macrophage colony-stimulating factor-induced proliferation and gene expression
Reference: Feldman GM, Rosenthal LA, Liu X., Hayes MP, Wynshaw-Boris A., Leonard WJ, Hennighausen
L., Finbloom
DS.: STAT5A-deficient mice demonstrate granulocyte-macrophage colony-stimulating factor-induced proliferation and gene expression. Blood 1997 Sep 1;90 (5):1768-1776.
Stat5a (+/-)
A targeting construct containing a 2.2-kb EcoRI-XbaI
fragment from the 5'-upstream region and a 5.0-kb XbaI-XbaI
fragment derived from the internal region of the Stat....
Stat5a (-/-)
A targeting construct containing a 2.2-kb EcoRI-XbaI
fragment from the 5'-upstream region and a 5.0-kb XbaI-XbaI
fragment derived from the internal region of the Stat....
Examples
for
application
of
K.O. mice
in medical
research
•
CFTR k.o. mice as a model for cystic fibrosis
•
Leptin
and LR k.o. mice as a model for obesity
•
Stefin
k.o. mice as a model for myoclonic epilepsy
•
SOCS2 k.o. mice as a moel
for high growth
Fusion
genes
(specific
promoter
+ reporter gene) can reveal
temporal
and
spatial
expression
profile
Mouse embryo expressing beta-galactosidase
under
control of the Oc2 gene promoter (knock-in)
Stat5a knock out phenotype
Stat5a is a member of the Stat super family of transcription factors. As a part of the Stat/Jak
pathway it regulates development of the ductal
and
alveolar system in the mammary gland during pregnancy and milk synthesis during lactation. Double Stat5a-/-
K.O. show clear underdevelopment of the
alveolar tissue.
Gene knock-in is a powerfull
tool for
gene expression
studies
Green fluorescent protein (GFP) expression under the control of the Tbx6 gene. This is a “knock-in”
of a histone-GFP fusion gene into the Tbx6 locus.
The fluorescence is a readout of Tbx6 gene activity
Lentivirus
delivery
of
shRNA
to mouse
embryos
Recently, a functional (sh)RNA
has
been introduced
into
mice using a lentivector. An H1promoter- driven shRNA
targeted against the
gene encoding green fluorescent protein (GFP) was introduced into one of the long terminalrepeats (LTRs) of a human immunodeficiency virus (HIV)-derived lentivector.
Owing to the
mechanism of reverse transcription, this
resulted in the pro-virus containing two
copies of the shRNA, one in each LTR.
This vector was introduced into fertilized
eggs that were generated from animals
known to contain a functional GFP
transgene. The reduction in GFP
fluorescence was observed in early
embryos and
in the pups born from
these eggs. From Clark and Whitelaw, 2003
Advantages and disadvantages of random transgene
integration
Advantages•
Short time to produce
•
High level of expression•
Simple vectors
•
No host genome sequence information required
•
Relatively cheap
Disadvantages•
Random integration
•
Multiple integration sites
•
No control over the copy number of the transgene
•
Alterations at integration site
•
Each animal has different genotype
Antisense
technology
was
an
early “forward
genetics”
tool
Napoli et al., 1990:Addition
of
the
gene coding
for
the
key
enzyme
for
flower pigmentation in
petunia
plants
did
not result
in darker flower
color. Surprisingly, many of the petunia plants carrying additional copies of this gene did not show the expected deep purple or deep red flowers but carried fully white or partially white flowers. When the scientists had a closer look they discovered that both types of genes, the endogenous and the newly introduced transgenes, had been turned off. The
phenomenon was first named "co-suppression of gene expression“.
A few years later plant virologists have
found
that plants carrying only short regions of viral RNA sequences not coding for any viral protein protected
plants
from
viral
infection.
They put also
short pieces of plant gene sequences into plant viruses. Indeed, after infection of plants with these modified viruses the expression of the targeted plant gene was suppressed. This phenomenon was
called
“virus-induced gene silencing”
(“VIGS”).
These phenomena
were
collectively called
post transcriptional
gene silencing
- PTGS.
History
The mex-3B
inactivation
causes embryonic
arrest.
a, Negative control b,
Embryo from uninjected
parent (showing normal pattern of endogenous mex-3
RNA). c, Embryo from a parent injected with purified mex-3B
antisense RNA. d,
Embryo from a parent injected with dsRNA
corresponding to mex-3B; no mex-
3
RNA is detected.
Fire
and
Mello, 1998: dsRNA
is the
causative
agent of
interference (RNAi)
HistoryGuo
and
Kemphues, 1995:
Injection
of
either
sense
or anti-sense
RNA into
C. elegans
interferes
with
specific
gene expression
Function of RNAi•
Supression
of
transposon
mobilisation
and
acumulation
of
repetitive
DNA in the germline.
•
Prevention
of
viral
infections.•
Natural
mechanism
for
the
regulation
of
endogenous
gene expression.
•
An excellent
tool
for
experimental
gene silencing
and
forward
genetics.
Primer recesivne
epistaze
črn: B/-;E/-
rjav:
b/b; E/-
zlat;
-/-;e/e
Potomstvo
dihibridnega
križanja
(B/b;E/e x B/b;E/e bi dalo
naslednje
fenotipsko
razmerje:
9 črnih3 rjavi4 zlati
Epistaza (Bateson, 1909)
Penetranca
Penetranca: heterogeni model
Epistaza
(Fischer, 1918)
Interaction=0
…following
a paper
by
Fisher
in 1918, the
term ‘epistatic’
has
been
generally
used in yet
another
different
sense
from
its
original usage. In Fisher’s 1918 definition, epistasis
refers
to a deviation
from
additivity
in the
effect
of
alleles
at different
loci
with
respect
to their
contribution
to a quantitative
phenotype. Epistasis
in the
Fisher
sense
is closer
to the
usual
concept
of
statistical
interaction: departure
from
a specific
linear
model describing
the
relationship
between
predictive
factors
(here
assumed
to be
alleles
at different
genetic
loci).
Robustness, redundancy
and
epistasisIn a non-robustsystem
with
no backup
for
the
phenotypic
property, the
first
mutation
strongly
impairs
the
phenotype; as a result, subsequent
mutations
no longer
do much
harm, whichresults
in positive
epistasis
(ε
> 0).
In a redundant
system
mutations
have
initially
mild
effects
on fitness. However, as the
number
of
mutationsincreases, all
redundant
elements
will
eventually
be
damaged
by
mutations
and
the
function
eventually
collapses, resulting
in negative epistasis
(ε
< 0).
Callipyge
–
polar
overdominance
The
mutation
responsible
for
the
callipyge
trait
is located
within
an
imprinted
gene cluster
on the
distal
end
of
ovine
chromosome
18. The
callipyge
phenotype
is inherited
in a non-Mendelian
mode termed
polar
Overdominance
in which
only
animals
that
inherit
a normal allele
(wild
type; +) from
the
dam and
the
mutant callipyge
allele
from
the
sire (CLPGPat) exhibit
the
callipyge
phenotype. Maternal
heterozygotes
(CLPGMat/+Pat) and
callipyge
allele
homozygotes
(CLPGMat/CLPGPat) have
muscling
and
carcass
compositions
that
are similar
to normal sheep
(wild
type
homozygotes; +/+).
Callipyge gene region
Six
known
transcripts
are indicated
along
with
the
direction
of
transcription
(arrows). Transcripts
expressed
from
the
paternal
allele
are shown
as orange
arrows
and
those
expressed
from
the
maternal
allele
are shown
as black
arrows. A blue
line indicates
the
position
of
the
causative
mutation. Alleles
with
an
A are wild
type
(+) and
the
mutant callipyge
allele
(CLPG) has
a G at this
position.
Mapping
of
PEG11 and
PEG11AS transcripts
In worms
and
in plants
an amplification
mechanism
is present, therefore
already tiny
amounts
of
siRNA
can trigger
RNAi.
Step 1.
Isoaltion
of
developing embryo at blastocyst
stage. In order
to make detection
of
chimeras
easier, we
can
use
embryos from
the
strain
with
black
fur
(C57),
Step 2.
ES cells
from
ICM are removed from
the
black-fur
blastocyst. Cells
are grown
in cell
culture
under
nondiferentiating conditions.
Step 3.
ES cells
are transfected
with
homologous
recombination
construct. Selection
for
homologous
recombinants
is performed
by
adding
neomycin
(G418)
and gancyclvir
to the
growth
medium.
+ neomycin + gancyclovir
Note: integration
of
neo cassette
provides
resistance
to neomycin
(G418) and absence
of
tk cassette
provides
insensitivity
to gancyclovir. The
selected
genotype
is neo+, tk-.
Step 4.
Homologously
recombined embryonic
stem cells are injected into a blastocyst
from
an
animal
with
white
fur.
•
Cultivation of ES cells enables genetic modifications, efficient selection and preserves totipotency
in vitro.
•
Microinjection of genetically modified ES cells into recipien
blastocysts
causes formation of chimeras.
Step 5.
Implantation
of
several chimeric
blastocysts
into pseudo-pregnant, white fur mouse, mated
with
vasectomized
white-fur
males.
This
system allows
us to distinguish
chimeric
and
wild
type
progeny
by
fur
colour.
Step 6.
Foster mother will give birth to a number of mice. Some will be normal wild type white-fur mice but others will be chimeric. Chimeric
mice have many of their cells from the original white-fur blastocyst
but some of their cells will be derived from recombinant stem cells. Fur cells from recombinant stem cells produce black patches which are easily detected.
Using
coat
colour
markers, chimerism can
easily
be
detected
phenotypically
•
Chimera
for
tyrosinase gene in epithelial
cells.
Brem et al., 1992
Step 7.
Mating of
chimeric
mice with wild-type white
fur mice
will
result
in a number
of recombinant
(black) and
some wild
type
(white) animals, provided
that
the gonads of the chimeric
mice are also
chimeric. The
proportion
of
recombinant
offspring
depends
on the prportion
of
recombinant
cells
in their
gonads. All
black
mice
are heterozygous
for
the homologous recombination.
Step 8.
Intermating
of heterozygous black mice (+/ H) will result in Mendelian
proportion 1:2:1 of wild type white, heterozygous black
and homozygous black offspring, respectively. In the test cross will heterozygotes
(+/ H) produce 50% wild type and 50% black progeny,
whereas homozygous recombinants (H
/ H) will produce 100% black progeny. The pure breeding mouse strain is a "knockout mouse".
knockout mouse
Test cross:
50% wt, 50% black
100% black
Efficacy of homologous recombination
Length of homologous sequences
Hom. Rec. Efficiency
Base pair
25bp 2000bp
Advantages of the MOUSE system
Life span: approx. 2.5 yearsGestation : 21 daysLitter size: 8 to 12 Generation time: three monthsA number
of
inbred and outbred
strains
Genomic databaseMost advanced
genetic technologies
Cost per mouse/
$2 to 24$Relative
low
housing
costs/animal
Over 90% gene sequence
identity
to manLarge enough for physiological studies
Pitfalls
of
constitutive homologous
recombination
•
Constitutive
gene inactivation
in all tissues
•
Not suitable
for
developmenally important
genes
(embryonic
lethality)
•
Systemic
effects
(knock-
in)•
Ectopic
expression
(knock-in)
•
Presence
of
selection
cassette
Site specific recombination:Cre-Lox system, Flp
recombination
From P1 phage:Cre recombinase
Lox site(approx 30 bp)
Excise and integrate DNA: knock
out
and
knock
in
Excisea b c
a bc
a c
b
Integratege
f
e f g
Cre/lox system
in vivo
-
Temporal and spatial targeting-
Knock-out
and
knock-in
-
Single point mutation-
Translocation
(double
targeting
event)
GutKO in the gut
onlyKO in the adult only
Adult-P
XLox mouse Cre
mouse
TissueTissue
specificspecific
gene gene inactivationinactivation CreCre/Lox /Lox knockknock
outout
technoogytechnoogy
Cell type specific Cell type specific promoterpromoter Cre Target geneTarget geneloxPloxP loxPloxP
X
Cre Cre
TargetTarget
tissuetissue AllAll
otherothertissuestissues
Data driven approach
•
Sequencing
projects•
SNP chip
approach
•
Transcriptomics•
Proteomics
•
Metabolomics•
Interactomics
State of the art -
tools•
Genetic markers
•
RH mapping panels
•
Mapping pedigrees
•
Large fragment genomic clones
•
cDNA
libraries
•
EST databases
•
Whole
genome sequence publicly
available
•
Microarrays for expression profiling
Genome Transplantation
in Bacteria: Changing
One Species
to Another
As a step toward
propagation
of
synthetic genomes, we
completely
replaced
the
genome of
a bacterial
cell
with
one from
another species
by
transplanting
a whole
genome as naked
DNA. Intact
genomic
DNA from Mycoplasma
mycoides
large
colony
(LC), virtually
free
of
protein, was
transplanted
into Mycoplasma
capricolum
cells
by
polyethylene glycol–mediated
transformation. Cells selected
for
tetracycline
resistance, carried
by the
M. mycoides
LC chromosome, contain
the complete
donor
genome and
are free
of detectable
recipient
genomic
sequences. These
cells
that
result
from
genome transplantation
are phenotypically
identical
to the
M. mycoides
LC donor
strain
as judged
by several
criteria.
Colony
hybridization
of
the
M. mycoides
LC (genome donor), M. capricolum
(recipient
cell), and
transplants
from
four
different
experiments
that
were
probed
with
a polyclonal
antibody
specificfor
the
M. capricolum
VmcE
and
VmcF
surface
antigens
or with
monoclonal
antibodies
specific
for
the
M. mycoides
LC VchL
surface
antigen.
Association studies
•
Search
for
association
between
phenotype and
genotype
(WGSc)
•
Identification
of
candidates
within
the target
regions
•
Single
locus
vs. haplotype
approach
QTL studies
•
More than
20 studies
were
performed
in order
to detect
lactation
associated
QTLs
•
Whole
genome scans
(MS, SNP)•
QTL intervals
fairly
large
(several
cM)
•
Detected
QTLs
are often
breed
specific•
QTL analysis
is affected
by
epistasis,
stratification, assortative
mating, environmental
effects
QTL- what does it mean?QTL
=
quantitative trait locus
Piece
of
DNA with
a
measurable effect
on
quantiative
trait
Classical linkage analysis
m M? ?
m x? x
M x? x
Classical
linkage
analysis
–
daughter
design
m M? ?
+400 lbs -400 lbs
m x? x
M x? x
Classical
linkage
analysis
–
daughter
design
m M? ?
+400 lbs -400 lbs
m xq x
M x
Q x
Classical
linkage
analysis
–
daughter
design
m Mq Q
Population-wide
association
Population-wide
association
Population-wide
association
Population-wide
association
•
Within
the
family
•
~10 Marker / Chromosom•
Microsatellites
m q
MQ
m Mq Q
Linkage
vs.
Population-wide
association
•
Within
the
population
•
1000s
of
Markers
/ Chromosome•
SNPs
Quantitative
genetics
approaches
•
Classical
linkage
analysis
within
the
family•
Population-wide
association
study
•
Search
for
associations
between
phenotype
and
genotype
m M
Selection signature•
Phenotypical
selection
on thait
A
•
Performance
improves
and
the
frequency
of
positive
allele
too
Perform
ance
Allelfrequency
Perform
ance
Allelfrequency
•
„extended
Haplotype Homozygosity“
(EHH)
•
Test statiscs
curve–
Non
mutated
allele
–
Mutated
allele
•
Surface
under
the
curve•
No selection
–
both
surfaces
equal•
Selection
–
surfaces
different•
Log ratio
Sabeti
et al. (2001) Voight
et al. (2006)
Test for
selection
signature
Milk trait
related
QTLs
in bovine
genome
1514165
TAMU, QTL viewer
Grisart et al., 2002
DGAT1 was
the
first
milk
trait
QTL confirmed
at molecular
level
Cohen-Zinder et al., 2005
QTL region on BTA6
QTL detection
in commercial
populations
“The only option to achieve the standard of rigorous proof for identification of a gene
underlying a QTL in commercial populations is to collect multiple pieces of evidence, no single one of which is convincing, but which
together consistently point to a candidate gene.”
Trudy F.C. Mackay, 2001
Večina gospodarsko pomembnih lastnosti je pogojena z velikim številom genov, fenotipske
vrednosti za te lastnosti pa so v populaciji normalno porazdeljene
X
n
X’
SD
meja
odbranih
srednja
vrednostodbranih
P
Večina gospodarsko pomembnih lastnosti je pogojena z velikim številom genov, fenotipske
vrednosti za te lastnosti pa so v populaciji normalno porazdeljene
X
n
Selekcija
•
Intenzivnost
selekcije
izražamo
z odstotkom
odbranih
živali
•
Intenzivnost
selekcije
je pogojena
z reprodukcijskimi
omejitvami
•
i=SD/P•
G = starost
osebkov
ko
imajo
spolno
zrele
potomce
Heritabilnost (h2)
•
h2
= VG/VP•
h2
je populacijski
parameter in ga
zato
določamo
v različnih
populacijah•
poprečne
ocene
h2
za
posamezne
lastnosti
so dovolj
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PV
•
VP. h2
=VG; P.h=G; h je regresijski koeficient
C to G transition
in the
-LG promoter reduces
AP-2 binding
in vitro
Lum et al., 1997
C to G transition
increases proportion
of
-LG in milk
Kuss et al., 2003
Lenasi et al., 2006
Alternative splicing
is common
in lactoprotein
genes
In vitro splicing
results
FN ex-1
gbpromoter
FN in+1
FN in-1
FN ex+1
FN EDA
PROMOTER EXON 1 INTRON 1
-casein inserts in the pSVEDATot vector
pSVEDATot
vector
Proposed
model of
co-transcriptional splicing
of
b-casein
mRNA
CTD
mRNA
CBC
pol RNA II
?SR
DNA
SPL
Polymorphism
i the
intron
1 is part of
the
nucleotide sequence, typical
for
ESE element
GAAGAA →
GAUGAA ; AAGAAAAU →
AUGAAAAU
OPERATIONAL GENOMICSOPERATIONAL GENOMICS
Breeding strategies
Technology Transfer Technology Transfer
Structural Genomics
Functional Genomics
Population Genomics
OPERATIONAL GENOMICSOPERATIONAL GENOMICS
Improved monitoring and surveillance
New intervention strategies (vaccination, drugs)
External & emerging policies
(avian
flu)
Technical evaluation of outputs
Standards for operational outcomes
EXAMPLES
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