1. Harlan Transgenic Services
Harlan UK & Harlan BV Transgenic Services platforms

2. 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)

3. 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

4. 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:

5. 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

6. 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 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,…)

7. A complete integrated transgenesis platform
Conditional targeting
Gene of interest
Targeting vector
ES cells electroporation and selection
Isolation and characterization of genomic clone
Conventional targeting
cDNA isolation
Vector design and construction
Selection cassette deletion
Production of homozygous recombinant ES cells
DNA sequencing
Microinjection of recombinant Cells and Generation of chimerae
Lentivirus production and infection
E. coli
Expression vector
Cloning vector
Genotyping
Heterozygous germ line transmission mice
Protein overexpression
Breeding service
Yeast
Macro-observation
Speed congenic
Sanitization
Proteomics
Homozygous mutant mice
Cryopreservation
Mammalian cells
Microarrays
Breeding with recombinase expressing mice
Phenotype analysis
Human pathology
Histopathology

8. 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

9. 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, …)

10. Step III Step V Step VI Step IV Step VII
A complete integrated transgenesis platform
Conditional targeting
Gene of interest
Targeting vector
ES cells electroporation and selection
Isolation and characterization of genomic clone
Conventional targeting
cDNA isolation
Vector design and construction
Selection cassette deletion
Production of homozygous recombinant ES cells
DNA sequencing
Microinjection of recombinant Cells and Generation of chimerae
Lentivirus production and infection
Step III
Genotyping
Heterozygous germ line transmission mice
Step III: Generation of Chimerae
Step V
Breeding service
Macro-observation
Step IV: Mice breeding
Speed congenic
Sanitization
Proteomics / metabolomics
Step V: genotyping
Homozygous mutant mice
Cryopreservation
Step VI: embryo services
Microarrays
Breeding with recombinase expressing mice
Phenotype analysis
Step VI
Step IV
Human pathology
Histopathology
Step VII

11. 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 1)
(Package 2)
(Package 3)
(Package 4)

12. 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 )

13. blastocyst Re-implantation (ICR-CD1 pseudopregnant female)
Positives clones
blastocyst Re-implantation (ICR-CD1 pseudopregnant female)
Cells Integration
in the embryo
Microinjection
Of ES cells
In host embryo (C57BL6)  
heterozygote
Homozygous
For the mutation  X X
WT “C57Bl” Black
Or ICR-CD1 
Chimeric mice

14. Microinjection of ES cells
Microinjection station

15. Microinjection of ES cells
C57BL6 blastocyst

16. Microinjection of ES cells
Client request
Information regarding the conditions of culture of the ES cell
Culture of the cells
Microinject into blastocysts
Freeze a part
Delivery of at least 3 chimerae OR
Microinjection of maximum 150 blastocysts
Stop

17. 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)

18. 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.

19. 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.

20. Sanitization – Embryos rederivation

21. Sanitization – Embryos rederivation Embryos re-implantation

22. 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

23. 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.

24. 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

25. Genotyping: the service
Client request No
Client PCR conditions
Obtain the DNA information
Yes
Client protocol transfer
PCR conditions setup
Not OK
PCR of samples 1 to 24 OK
Stop
PCR of samples up to 24

26. 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.

27. 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

28. Speedcongenic A B F1 B Mendel X + X + + +
Build a congenic strain quicker than by a classical backcross:
Take advantage of the variation of the mendelian statistic: A B F1 B
Mendel X + X + + +

29. Speedcongenic
50 % + 25 %

30. 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

31. Speedcongenic
Classical backcrossing
Speedcongenic F1
50 % N2
75 %
79-80 % N3
87,5 %
92-94 % N4
93,8 %
97-99 % N5
96,9 %
99,9 % …
N10
Speedcongenic: 5 generations in 1 year to obtain a congenic strain

32. 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.

33. Speedcongenic: marker-assisted selection
Balb/cJ
C57BL6/J
129X1/SvJ
D1MIT316
226
227
229
D1MIT430
127
119
123
D1MIT169
225
221
D1MIT215
167
159
151
D1MIT102 86 90
D1MIT159
146
206
194

34. Speedcongenic: the package
Obtain information (donor strain, host strain, localization of the mutation, PCR conditions and receive animals)
Client request
Breeding
DNA extraction
Heterozygous screening
Mouse House
Microsatellites analysis
Identification of the two best N+1 breeder
Send best N5 animals

35. 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.

36. 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

37. Step III Step V Step VI Step IV Step VII
A complete integrated transgenesis platform
Conditional targeting
Gene of interest
Targeting vector
ES cells electroporation and selection
Isolation and characterization of genomic clone
Conventional targeting
cDNA isolation
Vector design and construction
Selection cassette deletion
Production of homozygous recombinant ES cells
DNA sequencing
Microinjection of recombinant Cells and Generation of chimerae
Lentivirus production and infection
Step III
Genotyping
Heterozygous germ line transmission mice
Step III: Generation of Chimerae
Step V
Breeding service
Macro-observation
Step IV: Mice breeding
Speed congenic
Sanitization
Proteomics / metabolomics
Step V: genotyping
Homozygous mutant mice
Cryopreservation
Step VI: embryo services
Microarrays
Breeding with recombinase expressing mice
Phenotype analysis
Step VI
Step IV
Human pathology
Histopathology
Step VII