Harlan Transgenic Services Harlan UK Harlan BV Transgenic
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 Harlan. Europe with the Bio. Vallée transgenesis platform (Belgium)
Bio. Vallée 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
Bio. Vallée 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 Bio. Vallée 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 c. DNA isolation ES cells electroporation and selection 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 Expression vector Cloning vector Genotyping E. coli Heterozygous germ line transmission mice Protein overexpression Breeding service Macro-observation Speed congenic Proteomics Sanitization Homozygous mutant mice Cryopreservation Yeast Breeding with recombinase expressing mice Microarrays Phenotype analysis Mammalian cells Human pathology 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 c. DNA 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 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 Cryopreservation Breeding with recombinase expressing mice Microarrays Phenotype analysis Human pathology 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-CD 1 pseudopregnant female) Microinjection Of ES cells In host embryo (C 57 BL 6) Cells Integration in the embryo heterozygote Homozygous For the mutation X X WT “C 57 Bl” Black Or ICR-CD 1 Chimeric mice
Microinjection of ES cells Microinjection station
Microinjection of ES cells C 57 BL 6 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
Speedcongenic Build a congenic strain quicker than by a classical backcross: Take advantage of the variation of the mendelian statistic: F 1 B A B Mendel X X + +
Speedcongenic 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 F 1 50 % N 2 75 % N 2 79 -80 % N 3 87, 5 % N 3 92 -94 % N 4 93, 8 % N 4 97 -99 % N 5 96, 9 % N 5 99, 9 % … N 10 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 c. M are tested during the process.
Speedcongenic: marker-assisted selection Balb/c. J C 57 BL 6/J 129 X 1/Sv. J D 1 MIT 316 227 229 D 1 MIT 430 127 119 123 D 1 MIT 169 225 221 D 1 MIT 215 167 159 151 D 1 MIT 102 86 86 90 D 1 MIT 159 146 206 194
Speedcongenic: the package Client request Breeding Obtain information (donor strain, host strain, localization of the mutation, PCR conditions and receive animals) DNA extraction Mouse Heterozygous screening Microsatellites analysis Identification of the two best N+1 breeder Send best N 5 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 C 3 H) • 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 c. DNA 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 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 Cryopreservation Breeding with recombinase expressing mice Microarrays Phenotype analysis Human pathology Histopathology Step VII Step IV Step VI
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