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