Harlan Transgenic Services Harlan UK & Harlan BV Transgenic Services platforms
Jan 15, 2016
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
Gene of interest
Isolation and characterization of genomic clone
cDNA isolation
DNA sequencing
Vector
AP r
ALPHA
P(BLA)
P(LAC)
ORI
Vector design and construction
Expression vector Cloning vector
Protein overexpression E. coli
Yeast
Mammalian cells
Conditional targeting
ES cells electroporation and selectionTargeting vector
Conventional targeting
Selection cassette deletion
Production of homozygous recombinant ES cells
Microinjection of recombinant Cells and Generation of chimerae
Heterozygous germ line transmission mice
Breeding service
Homozygous mutant mice
Breeding with recombinase expressing mice
Speed congenic
Cryopreservation
Sanitization
Genotyping
Phenotype analysis
Macro-observation
Histopathology
Microarrays
Proteomics
Human pathology
Lentivirus production and infection
The complete offer
• Design and construction of vectorsTargeting vectors/Lentiviral vectorsExpression vectorsCloning vectors
• Cell cultureES cells culture (transgenesis)Eukaryote cells cultureStable transfection in eukaryote cells (overexpression of proteins)Production of lentivirus
• GenotypingSouthern BlotPCR (detection on agarose gel)Genome scanning (speed congenic, genetic background identification)SNP/microsatellite detectionZygosity test
The complete offer
• Animal facilityMice breeding (managed by Harlan)Generation of chimera (microinjection/aggregation/lentiviral infection)Production of VAS malesEmbryo rederivationEmbryo cryopreservation (01/2008)
•Transgenic mice phenotypingMacro-observationHistopathological analysisMolecular analysis (Northern blot, RT-PCR, microarrays, proteomic, …)
A complete integrated transgenesis platform
Gene of interest
Isolation and characterization of genomic clone
cDNA isolation
DNA sequencing
Vector
AP r
ALPHA
P(BLA)
P(LAC)
ORI
Vector design and construction
Conditional targeting
ES cells electroporation and selectionTargeting vector
Conventional targeting
Selection cassette deletion
Production of homozygous recombinant ES cells
Microinjection of recombinant Cells and Generation of chimerae
Heterozygous germ line transmission mice
Breeding service
Homozygous mutant mice
Breeding with recombinase expressing mice
Speed congenic
Cryopreservation
Sanitization
Genotyping
Phenotype analysis
Macro-observation
Histopathology
Microarrays
Proteomics / metabolomics
Human pathology
Lentivirus production and infection
Step III
Step III: Generation of Chimerae
Step IV
Step IV: Mice breedingStep V
Step V: genotyping
Step VI
Step VI: embryo services
Step VII
The new services offer
Step III: Generation of Chimerae
• Microinjection of ES cells into blastocyst
• Lentivirus mice production (under development, 06/2008)
Step IV: Mice breeding: mouse house in Harlan facilities
Step V: Genotyping
• Mouse genotyping
• Speedcongenic
• Genetic monitoring
Step VI: Embryos services
• Sanitization
• Embryos Cryopreservation (Package 5, under development (01/2008))
(Package 2)
(Package 3)
(Package 1)
(Package 4)
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 )
Microinjection Of ES cells
In host embryo (C57BL6)
Cells Integration in the embryo
blastocyst Re-implantation (ICR-CD1 pseudopregnant
female)
Chimeric mice
XX
heterozygote
HomozygousHomozygous For the mutation
WT “C57Bl” BlackOr ICR-CD1
Positives clones
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
Freeze a partMicroinject into blastocysts
Delivery of at least 3 chimerae Microinjection of maximum 150 blastocysts
Stop
OR
Sanitization – Embryos rederivationPackage 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 rederivationEmbryos re-implantation
Sanitization – Embryos rederivation
Client request
Heterozygous Homozygous
Need at least 2 males and 10 females
Need at least 2 males and host strain
Colony amplification
Breeding and embryos transfer
Send at least 3 breeding pairs to the client
Health status of the recipient mothers as best guarantee
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.
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
Genotyping: the service
Client request
Client PCR conditions
PCR conditions setupClient protocol transfer
PCR of samples 1 to 24
PCR of samples up to 24
Obtain the DNA information
Stop
Yes
No
Not OK
OK
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
50
75
87,5
93,896,9 98,4 99,2 99,6 99,8 99,9
0
10
20
30
40
50
60
70
80
90
100
N1 N2 N3 N4 N5 N6 N7 N8 N9 N10
Generation
Ho
st p
ou
rcen
tag
e
Speedcongenic
A B
X
F1 B
XMendel
+
+
+ +
Build a congenic strain quicker than by a classical backcross:
Take advantage of the variation of the mendelian statistic:
Speedcongenic
0
20
40
60
80
100
120
65-67 68-70 71-73 74-76 77-79 80-82 83-85
% of host genome in N2
nu
mb
er
of
mic
e
50 % + 25 %
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 Obtain information (donor strain, host strain, localization of the mutation, PCR conditions and receive animals)
BreedingHeterozygous screeningDNA extraction
Mouse House 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
Gene of interest
Isolation and characterization of genomic clone
cDNA isolation
DNA sequencing
Vector
AP r
ALPHA
P(BLA)
P(LAC)
ORI
Vector design and construction
Conditional targeting
ES cells electroporation and selectionTargeting vector
Conventional targeting
Selection cassette deletion
Production of homozygous recombinant ES cells
Microinjection of recombinant Cells and Generation of chimerae
Heterozygous germ line transmission mice
Breeding service
Homozygous mutant mice
Breeding with recombinase expressing mice
Speed congenic
Cryopreservation
Sanitization
Genotyping
Phenotype analysis
Macro-observation
Histopathology
Microarrays
Proteomics / metabolomics
Human pathology
Lentivirus production and infection
Step III
Step III: Generation of Chimerae
Step IV
Step IV: Mice breedingStep V
Step V: genotyping
Step VI
Step VI: embryo services
Step VII