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

20092009

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

qq

Qq

QQ

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

zanesljive, da

jih

lahko uporabimo

v ocenjevanju

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