Transcript Harlan Presentation Template

Harlan Transgenic Services
Harlan UK & Harlan BV Transgenic Services platforms
Harlan Transgenic Services
One existing platform in UK:
• Embryos rederivation
• Embryos cryopreservation
• Genotyping
• Health monitoring
Increase the services panel and capacity:
Association of HarlanEurope with the BioVallée transgenesis
platform (Belgium)
BioVallée
Non-profit organization funded with the support of the Walloon
region and the EU
Capacity:
• Transgenic development (vegetal and animal),
• Genetic analysis (DNA and RNA),
• Proteomics
• Metabolomics
Goal: Create economic value in the Walloon region
BioVallée
Creation of two spin-offs: Delphigenetics (DNA engineering) and
DNAVision (Genetic Analysis)
Incorporation of a new company in December:
BV Transgenic Services: association between Harlan and the
BioVallée Transgenesis Platform
Creation of a new Harlan Services Platform:
BV Transgenic Services capacity
Team involved in the development of animal models from the DNA
construct to the phenotype analysis
Access to technologic platforms:
• Molecular biology laboratory
• SPF animal facility (with IVC racks)
• Genetic analysis platform
• Phenotyping platforms: Proteomics, microarrays, metabolomics,
histo-pathology
• Cell culture
BV Transgenic Services capacity
Example of equipment:
• 2 Affymetrix Gene Chip Expression/High-throughput SNPs platforms
Possibility to use custom microarrays
• 1 Real-time RT-PCR system for microarray result validation and/or
low-throughput screening (ABI 7900 RTPCR)
• 1 Bioanalyseur Agilent 2100 for RNA-quality assessment.
• 1 Pyrosequencing machine
• 1 ABI 16 capillary genetic genotyper
• 1 ABI 48 capillary genetic genotyper /sequencer
• 1 DNA threshold system
• 4 PTC-100 and - 200 PCR systems
• Biostatistics and Bioinformatics for microarray and RT-PCR analyses
(clustering, parametric and non-parametric tests, multiple testing
correction, class comparison, class prediction …), and interpretation
(in term of pathways,…)
A complete integrated transgenesis platform
Conditional targeting
Targeting vector
Gene of interest
Conventional targeting
Isolation and characterization of genomic clone
cDNA isolation
ES cells electroporation and selection
Vector design and construction
Selection
cassette
deletion
Production of homozygous
recombinant ES cells
P(BLA)
ALPHA
P(LAC)
DNA sequencing
AP r
Vector
Microinjection of recombinant Cells and Generation of chimerae
ORI
Lentivirus production and infection
Expression vector
Cloning vector
Genotyping
E. coli
Heterozygous germ line transmission mice
Protein overexpression
Breeding service
Macro-observation
Speed congenic
Proteomics
Homozygous mutant mice
Yeast
Phenotype analysis
Human pathology
Cryopreservation
Breeding with recombinase
expressing mice
Microarrays
Mammalian cells
Sanitization
Histopathology
The complete offer
• Design and construction of vectors
Targeting vectors/Lentiviral vectors
Expression vectors
Cloning vectors
• Cell culture
ES cells culture (transgenesis)
Eukaryote cells culture
Stable transfection in eukaryote cells (overexpression of proteins)
Production of lentivirus
• Genotyping
Southern Blot
PCR (detection on agarose gel)
Genome scanning (speed congenic, genetic background identification)
SNP/microsatellite detection
Zygosity test
The complete offer
• Animal facility
Mice breeding (managed by Harlan)
Generation of chimera (microinjection/aggregation/lentiviral infection)
Production of VAS males
Embryo rederivation
Embryo cryopreservation (01/2008)
•Transgenic mice phenotyping
Macro-observation
Histopathological analysis
Molecular analysis (Northern blot, RT-PCR, microarrays, proteomic, …)
A complete integrated transgenesis platform
Conditional targeting
Targeting vector
Gene of interest
ES cells electroporation and selection
Conventional targeting
Isolation and characterization of genomic clone
cDNA isolation
Vector design and construction
Selection
cassette
deletion
Production of homozygous
recombinant ES cells
P(BLA)
ALPHA
P(LAC)
DNA sequencing
AP r
Vector
Microinjection of recombinant Cells and Generation of chimerae
ORI
Lentivirus production and infection
Step III
Genotyping
Step III: Generation of Chimerae
Macro-observation
Step IV: Mice breeding
Proteomics / metabolomics
Step V: genotyping
Step VI: embryo services
Step V
Heterozygous germ line transmission mice
Breeding service
Speed congenic
Sanitization
Homozygous mutant mice
Breeding with recombinase
expressing mice
Microarrays
Phenotype analysis
Human pathology
Cryopreservation
Histopathology
Step VII Step IV
Step VI
The new services offer
Step III: Generation of Chimerae
• Microinjection of ES cells into blastocyst (Package 1)
• Lentivirus mice production (under development, 06/2008)
Step IV: Mice breeding: mouse house in Harlan facilities
Step V: Genotyping
• Mouse genotyping (Package 2)
• Speedcongenic (Package 3)
• Genetic monitoring
Step VI: Embryos services (Package 4)
• Sanitization
• Embryos Cryopreservation (Package 5, under development (01/2008))
Microinjection of ES cells(Package 1)
Access to genetically modified ES cells bank ( ex: IGTC).
Goal of the service: provide a genetically modified animal from
a “blast” (screen between the sequence of the gene of interest
and the genetically modified ES cells bank )
Positives clones
blastocyst Re-implantation
(ICR-CD1 pseudopregnant
female)
Microinjection
Of ES cells
In host embryo (C57BL6)
Cells Integration
in the embryo


heterozygote
Homozygous
For the mutation

X
X

WT “C57Bl” Black
Or ICR-CD1
Chimeric
mice
Microinjection of ES cells
Microinjection station
Microinjection of ES cells
C57BL6 blastocyst
Microinjection of ES cells
Client request
Information regarding the conditions
of culture of the ES cell
Culture of the cells
Microinject into blastocysts
Delivery of at least 3 chimerae
Freeze a part
OR Microinjection of maximum
150 blastocysts
Stop
Sanitization – Embryos rederivation
Package 4
Goal: to clean dirty animals (non pathogen free) to be
able to use it in a SPF environment
No possibility to clean live animals
Uterus is a pathogen free environment (at least for
the pathogen described by the FELASA)
Sanitization – Embryos rederivation
Two possibilities: hysterectomy or embryo transfer
Hysterectomy: take the pups just before birth cleanly and give it
to a foster mother.
Risk: foster refusal and contamination during the hysterectomy
(virus, MHV by example)
Embryo transfer: Take the embryos before the implantation
stage, wash it in sterile medium and reimplant it into a
pseudopregnant female.
This is the safest way to decontaminate a strain.
Sanitization – Embryos rederivation
Embryos collection:
The time mating in rodents
can easily be identified by
the vaginal plug that leave
the male after the act.
That’s also the mating that
induce the ovulation and
the hormonal changes
necessary to the
preparation of the uterus
to receive embryos.
Sanitization – Embryos rederivation
Sanitization – Embryos rederivation
Embryos re-implantation
Sanitization – Embryos rederivation
Client request
Heterozygous
Homozygous
Colony amplification
Need at least 2 males
and host strain
Need at least 2 males
and 10 females
Breeding and embryos transfer
Health status of the recipient
mothers as best guarantee
Send at least 3 breeding pairs to the client
Genotyping (Package 2)
Detection of genetic modifications by PCR
The key factor of a good genotyping result is to
begin by a good PCR protocol setup (primers
design, best buffer and best PCR cycle).
That’s the first obligatory step of our genotyping
service (protocol transfer or complete setup based
on the DNA sequence). Our goal is to detect the WT
and the mutant in the same reaction tube.
Genotyping: the service
The package: different possibilities:
• Setup of PCR protocol
• Transfer of client protocol
• DNA extraction
• Genotyping of 1 to 24 samples
• Genotyping of more than 24 samples
Genotyping: the service
Client request
Client PCR conditions
No
Obtain the DNA information
Yes
Client protocol transfer
Not OK
Stop
OK
PCR conditions setup
PCR of samples 1 to 24
PCR of samples up to 24
Speedcongenic (package 3): introduction
Congenic strains or co-isogenic strains are genetically identical
except for a local part of their genome defined by a mutation, an
allele or a locus.
The development of such strains sharing the same genetic
background allows to study the effects of mutations in avoiding
the artifacts due to a unknown, a variable or a mixed genetic
background.
Speedcongenic : classic backcross
Goal:
Transfer a mutation from a genetic background to another.
Transfer a mutation from a donor strain to a host strain.
At least 10 generations, 2 to 3 years
100
99,8
99,6
99,2
98,4
96,9
99,9
93,8
90
87,5
80
75
Host pourcentage
70
60
50
50
40
30
20
10
0
N1
N2
N3
N4
N5
N6
Generation
N7
N8
N9
N10
Speedcongenic
Build a congenic strain quicker than by a classical backcross:
Take advantage of the variation of the mendelian statistic:
F1
B
A
B
Mendel
X
X
+
+
+
+
Speedcongenic
120
50 % + 25 %
number of mice
100
80
60
40
20
0
65-67
68-70
71-73
74-76
77-79
% of host genome in N2
80-82
83-85
Speedcongenic: marker-assisted selection
Mice strains differ genetically. Some genes are different (allele)
but differences non coding regions are more available and
informative to discriminate strains
We use genetic markers to differentiate strains in the
speedcongenic process.
We use microsatellites or SNPs (highest availability in the
genome and highest variability between strains even
substrains).
Microsatellites are small sequences present all along the
genome that can be amplified by PCR with the same primers
pair and can produce different sizes amplicons between
strains.
We are talking about polymorphism
Speedcongenic
Classical backcrossing
Speedcongenic
F1
50 %
F1
50 %
N2
75 %
N2
79-80 %
N3
87,5 %
N3
92-94 %
N4
93,8 %
N4
97-99 %
N5
96,9 %
N5
99,9 %
…
N10
99,9 %
Speedcongenic: 5 generations in 1 year to obtain a congenic strain
Speedcongenic: marker-assisted selection
At each generation, we genotype weaned animals to detect
heterozygotes.
The genome of these animals is then analysed for the
microsatellites chosen for their polymorphism between the two
strains of interest (host and donor strain).
The animal that possesses the most of host microsatellites is
used as founder for the next generation.
100 microsatellites spaced approximately by 15 cM are tested
during the process.
Speedcongenic: marker-assisted selection
Balb/cJ
C57BL6/J
129X1/SvJ
D1MIT316
226
227
229
D1MIT430
127
119
123
D1MIT169
225
221
221
D1MIT215
167
159
151
D1MIT102
86
86
90
D1MIT159
146
206
194
Speedcongenic: the package
Client request
Breeding
Obtain information (donor strain, host
strain, localization of the mutation, PCR
conditions and receive animals)
DNA extraction
Mouse House
Heterozygous screening
Microsatellites analysis
Identification of the two best N+1 breeder
Send best N5 animals
Genetic monitoring
Analysis of the genome of mice to identify a genetic
contamination or to confirm the inbred status of the strain.
Are you sure about the genome of your animal?
Are you really working with congenic strains?
Is the control animal that you use the best one?
These questions are important to ask before to begin an
experiment and to avoid unexpected results or bad conclusions.
Genetic monitoring
•The quick scan (analysis of 20 microsatellites)
Analysis of large colonies to confirm the absence of
genetic contamination,
Confirm the genetic background of a strain (do I work
with a CBA or a C3H)
• The genome scan (analysis of 100 microsatellites)
Identify a genetic contamination
Confirm the inbred status after backcrossing
• The genome scan + (analysis of 5000 SNPs)
Allow the discrimination between 20 strains
Sharp analysis of the genome
Useful in positional cloning
A complete integrated transgenesis platform
Conditional targeting
Targeting vector
Gene of interest
ES cells electroporation and selection
Conventional targeting
Isolation and characterization of genomic clone
cDNA isolation
Vector design and construction
Selection
cassette
deletion
Production of homozygous
recombinant ES cells
P(BLA)
ALPHA
P(LAC)
DNA sequencing
AP r
Vector
Microinjection of recombinant Cells and Generation of chimerae
ORI
Lentivirus production and infection
Step III
Genotyping
Step III: Generation of Chimerae
Macro-observation
Step IV: Mice breeding
Proteomics / metabolomics
Step V: genotyping
Step VI: embryo services
Step V
Heterozygous germ line transmission mice
Breeding service
Speed congenic
Sanitization
Homozygous mutant mice
Breeding with recombinase
expressing mice
Microarrays
Phenotype analysis
Human pathology
Cryopreservation
Histopathology
Step VII Step IV
Step VI