Caenorhabditis elegans .pdf



Nom original: Caenorhabditis_elegans .pdf
Auteur: MOHAMED

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Caenorhabditis elegans et la
génomique fonctionelle
Belhocine mohamed
Mrbelhocine.mohamed@gmail.com

Centre d’Immunologie de Marseille-Luminy

Model systems

Yeast

Drosophila

Arabidopsis

Phylogenetic tree

Evolution in genomics
ics
Yeast

Invertebrate m odels

M am m als

Caenorhabditis elegans
A

C

muscle

pseudocoelom

intestine

gonad

250 µm

B

mouth
pharynx intestine

anus

vulva
gonad

hypodermis

nerve chord

C. elegans : a model organism

• Mechanisms of apoptosis
• Neuronal function and development
• Intracellular signalling pathways (let-60 ras...)

Why C. elegans?

image taken from http://www.Devgen.nl

Small size
Transparent
Rapid generation time

0.1 mm

image taken from http://www.Devgen.nl

Small size
Transparent
Rapid generation time
Long-term frozen storage

0.1 mm

image taken from http://www.Devgen.nl

Small size
Transparent
Rapid generation time
Long-term frozen storage
Invariant and identified cell lineages

0.1 mm

P0

P1 AB

www.wormatlas.org

www.wormatlas.org

www.wormbase.org

image taken from http://www.Devgen.nl

Small size
Transparent
Rapid generation time
Long-term frozen storage
Invariant and identified cell lineages
Internally self-fertilising
Clonal

0.1 mm

image taken from http://www.Devgen.nl

Small size
Transparent
Rapid generation time
Long-term frozen storage
Invariant and identified cell lineages
Internally self-fertilising
Clonal

Genetic map

0.1 mm

Genetic map

unc-32
sma-2
LGIII

emb-24

lin-9

lin-12

0

6 chromosomes :
LGI, LGII, LGIII, LGIV, LGV, LGX
LG = Linkage group

0,01 cM

image taken from http://www.Devgen.nl

Small size
Transparent
Rapid generation time
Long-term frozen storage
Invariant and identified cell lineages
Clonal
Internally self-fertilising
Genetic map
Genomic sequence

0.1 mm

Physical map

The C. elegans genome
Genome size
Megabases (Mb)

C. elegans
Human

Drosophila
Yeast (S. cerevisiae)

97 Mb
3000 Mb
160 Mb
12 Mb

*Protein coding genes

42% of C. elegans genes have homologues in other species
60% of these genes are represented by EST

Estimated number
of genes*
20000
23000
13000
6000

Small size
Transparent
Rapid generation time
Long-term frozen storage
Invariant and identified cell lineages
Clonal
Internally self-fertilising
Genetic map
Genomic sequence
Easy trangenesis

Micro-injection of C. elegans

Plasmids
Phages
Cosmids
YACs

Transgenesis : 1 week
injection
DNA

wt

F1

F2

wt;Ex(DNA)

wt;Ex(DNA)

wt

No integration in the genome. Inheritance is non-Mendelian

Worm gene
Promoter

X

Cloning vector

GFP

1 mm

Embryonic expression of punc-53::GFP
A
early

P

late

P

A

A

dc

vc

dc

image taken from http://www.Devgen.nl

Small size
Transparent
Rapid generation time
Long-term frozen storage
Invariant and identified cell lineages
Clonal
Internally self-fertilising
Genetic map,
Genomic sequence
Easy trangenesis

Positional cloning
Saturating mutagenesis

0.1 mm

The Genetic Approach
(“Forward Genetics”)
Mutagenesis

Chemical Mutagens

e.g. EMS

Physical mutagens

e.g. X-rays

The Genetic Approach
(“Forward Genetics”)
Mutagenesis
Screen for the phenotype of interest
Selection of a mutant
Cloning of the gene

sma-2

unc-32
pat-2
emb-24
lin-9

lin-12
0.01

0

T05B2
ZK637

ZK637.8

10 kb

ZK637.9

ZK637.10
1 kb

ZK637.7

plasmid
AIE5
E5-lin-9
E5∆Pac

ZK637.11

rescue

+
+

-

Mutagenesis by heterologous transposition
Extrachromosomal array

..

..

..
transposase

Extrachromosomal array
X

Strain EN547
(+ ; oxEx166)

Mos1
Strain EG1470
(+ ; oxEx229)

Extra array
Mos1

Insertional mutation
Cloning by inverse PCR

Inverse PCR
Worm lysis

Enzymatic digestion
Sau3A
A’

A

5’

3’

Mos1

3’

B

5’

B’

Sau3A

Ligation
Sau3A
A’

A

A

Mos1
B

B
B’

Sau3A

1st reaction

Sau3A

Sau3A
A’

Sau3A
A’

B’

2nd reaction

Sequence with A ’
Analyse by comparison
with sequences in data base
Determination of the mutation

image taken from http://www.Devgen.nl

Small size
Transparent
Rapid generation time
Long-term frozen storage
Invariant and identified cell lineages
Clonal
Internally self-fertilising
Genetic map,
Genomic sequence
Easy trangenesis

Positional cloning
Saturating mutagenesis
Good bioinformatics (Acedb)
0.1 mm

Topomap
Microarray data

Correlation matrix

Scatter plot

Topomap

Kim et al. Science 2001




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