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ES cells—gene targeting

The confluence of two groundbreaking technologies led to the first gene-targeted mouse models: plasticity of embryonic stem cells that contribute to the germline¹,² and successful homologous recombination³,⁴,⁵ between the ES cell genome and an introduced DNA sequence typically known as a targeting vector. Gene targeting has been applied to understand mammalian physiology, to study human disease mechanisms, and pervaded biomedicine to create precision therapeutics.  NovoHelix provides a variety of options for engineering the desired genetic modification into your model. Our full service projects include the entire mouse model generation process from scoping out the design strategy and targeting vector construction to delivery of the mutant animal model. We also provide options to clients for subcontracting certain experimental workflows or phases of the project to us and many of these service descriptions are provided under our Support Services tab. A detailed cost estimate can be developed based on your specific project through our online Quote Request.

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model generation

Service

Catalog Nr

Service Description

Timeline

Deliverables

Pricing

  • Full Service 129P2/Ola Gene Targeting Project
  • vector construction, ES cell targeting and screening, chimera generation
 ESGT001
 129P2/OlaHsd - genotype Aw/Aw Oca2p Tyrc-ch/Oca2p Tyrc-ch

 ~7 - 8 weeks from time of blastocyst injection. 

 2 gene targeted founders or 50 liveborn pups (if expression of the targeted allele would lead to embryonic or perinatal lethality) whichever comes first

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  • Full Service C57BL/6N Gene Targeting Project
  • vector construction, ES cell targeting and screening, chimera generation 
 ESGT002

 C57BL/6 strain transgene pronuclear microinjection

 ~7 - 8 weeks from time of blastocyst injection. 

 2 gene targeted founders or 50 liveborn pups (if expression of the targeted allele would lead to embryonic or perinatal lethality) whichever comes first

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  • Full Service BALB/cJ Gene Targeting Project
  • vector construction, ES cell targeting and screening, chimera generation 
 ESGT003
 C57BL/6 strain transgene pronuclear microinjection

 ~7 - 8 weeks from time of blastocyst injection. 

 2 gene targeted founders or 50 liveborn pups (if expression of the targeted allele would lead to embryonic or perinatal lethality) whichever comes first

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  • KOMP ES cell validation and injection package
  • Southern blot validation and blastocyst injection for chimera generation 
 ESGT004

 C57BL/6 strain transgene pronuclear microinjection

 ~7 - 8 weeks from time of blastocyst injection. 

 2 gene targeted founders or 50 liveborn pups (if expression of the targeted allele would lead to embryonic or perinatal lethality) whichever comes first

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 ES Cell Gene Targeting (129P2/Ola ES Cells)
 ESGT006
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 ES Cell Gene Targeting (C57BL/6N ES Cells)
 ESGT007
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 ES Cell Gene Targeting (BALB/cJ ES Cells) 
 ESGT008
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support services

Service

Catalog Nr

Service Description

Timeline

Deliverables

Pricing

 Gene Targeting Vector Construction
 ESGT005
 Isogenic gene targeting vector construction and Sanger-sequence verified.

We will provide an in silico map and provide restriction fingerprinting for plasmid structure via 3 digests. DNA is purified by a propriety AEX (anion exchange) chromatography and suitable for microinjection into mouse zygotes or nucleofection/electroporation.  The DNA is dialyzed on a membrane support in microinjection or electroporation buffer and then spun to remove particulate matter to obviate microinjection needle clogging.

 1- 4 weeks depending on targeting vector complexity

 5-10 micrograms of isogenic gene targeting plasmid; sequence validation, in silico map and 3  digests for restriction fingerprinting overall structure. 

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  • ES cell 2nd Electroporation 
  • 129P2/Ola ES Cells
  • Cre/Flpo
 ESGT009
Service package includes simple loxP or frt site-flanked selection cassette removal or lox-stop-lox removal with Cre/Flpo.  While NovoHelix is equipped with the ability to deploy site-specific recombinases Cre/Flpo/Dre/SCre/VCre/Vika/Nigri/Panto, please review SSR pages for more information regarding these tools and more complex recombinase technologies.

2 - 4 weeks

3 validated clones post excision

  • ES cell 2nd Electroporation 
  • C57BL/6N ES Cells
  • Cre/Flpo
 ESGT010
 Service package includes simple loxP or frt site-flanked selection cassette or lox-stop-lox removal with Cre/Flpo.  While NovoHelix is equipped with the ability to deploy site-specific recombinases Cre/Flpo/Dre/SCre/VCre/Vika/Nigri/Panto, please review SSR pages for more information regarding these tools and more complex recombinase technologies. 

2 - 4 weeks

3 validated clones post excision

  •  ES cell 2nd Electroporation 
  • BALB/cJ ES Cells
  • Cre/Flpo
 ESGT011
 Service package includes simple loxP or frt site-flanked selection cassette or lox-stop-lox removal with Cre/Flpo.  While NovoHelix is equipped with the ability to deploy site-specific recombinases Cre/Flpo/Dre/SCre/VCre/Vika/Nigri/Panto, please review SSR pages for more information regarding these tools and more complex recombinase technologies.

2 - 4 weeks

3 validated clones post excision

  • ES cell 2nd Electroporation 
  • NOD/SCID-Il2rg-null ES Cells
  • Cre/Flpo
 ESGT012
 Service package includes simple loxP or frt site-flanked selection cassette or lox-stop-lox removal with Cre/Flpo.  While NovoHelix is equipped with the ability to deploy site-specific recombinases Cre/Flpo/Dre/SCre/VCre/Vika/Nigri/Panto, please review SSR pages for more information regarding these tools and more complex recombinase technologies. 

2 - 4 weeks

3 validated clones post excision

 ES cell screening by long-range PCR and Southern blot (NovoHelix-produced vector)
 ESGT013
 ES cell screening by PCR and Southern blot (client-provided vector)
 ESGT014
  • ES cell expansion and preparation for microinjection
  • service includes up to 3 clones; client-provided ES cells without feeders 
 ESGT015
  •  ES cell expansion and preparation for microinjection
  • service includes up to 3 clones as recommended for KOMP/EUCOMM cells or
  • client-provided ES cells feeder-dependent 
ESGT016
  • Blastocyst microinjection 
  • C57BL/6 blastocysts
ESGT017
  • Blastocyst microinjection 
  • C57BL/6-albino blastocysts
 ESGT018
Pathogen + Mycoplasma testing of client-provided ES cell lines 
ESGT019
Mycoplasma testing of client-provided ES cell clones 
 ESGT020

 NovoHelix provides mycoplasma testing using a multiplex 16S rRNA gene assay to broadly determine adventitious mycoplasma species infection.  This service fee is per mouse ES cell (mESC) clone. The assay detects over 60 species of Mycoplasma, Acholeplasma, Spiroplasma and Ureaplasma including the eight species most likely to afflict cell cultures: M. arginini, M. fermentans, M. hominis, M.hyorhinis, M. orale, M. pirum, M. salivarium, and A. laidlawii. 

Chromosome counting 
 ESGT021
Chimera breeding for germline transmission 
ESGT022
 Chimera Flpo breeding for germline transmission and selection cassette (Neo, Hygro, Puro, Bsd, Zeo, ClonNAT) removal
ESGT023
 Southern blot (NovoHelix-produced vector)
 ESGT024
Southern blot (client-provided vector)
ESGT025
Southern blot - probe design
ESGT026
Tetraploid embryo / ES cell Aggregation
ESGT027
Tetraploid embryo/ ES or iPSC cell aggregation includes amplification and freezing of clonal stocks, chromosome counting, mycoplasma testing, 2-cell embryo collection, electrofusion of 2-cell embryos to tetraploid embryos, overnight culture, removal of zona pellucida, aggregation with targeted ES cells (2 times per clone) or 2 iPSC clones, overnight culture, transfer of the ES cells/tetraploid aggregated blastocysts to pseudopregnant female.  A minimum of 2 chimeras are almost completely ES cell derived will be produced.  C57BL6 and 129 Svev mice will be provided for germline transmission tests.  Additional injection will be charged at $5,500 per session. Note use of ES cells or iPSCs have varying potentials to produce chimeras and/or confer germline transmission (GLT).
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 Derivation of mouse embryonic stem cell line
ESGT028

 Re-derive a ES cell line from mouse blastocysts, including ESC karyotype and sex determination. All mESC lines are derived under 2i or 3i media.  We provide service of mouse ES cell lines derivation from the strain of choice or background of your mouse line.

Mouse colony husbandry & management
 ESGT029
 mouse colony management including breeding, weaning, sampling, monitoring, per cage per day
Genotyping Assay Development - targeting vectors
ESGT030

 A genotyping strategy is provided for the removal of the selection cassette and an additonal strategy to monitor the targeted allele

 Purification & validation of client transgene for microinjection 
ESGT031
Purification of supercoiled plasmid by a proprietary anion exchange (AEX) chromatography, validation by restriction digestion and analysis by gel electrophoresis. Endotoxin levels are generally very low (0.05  – 0.5 ng LPS/µg) via our proprietary extraction method and are adequate for sensitive applications such as zygotic microinjection of mammalian embryos. The plasmid is predominantly in its supercoiled topology and free of RNA and protein contamination.  Linearization with a restriction endonuclease or with a homing nuclease such as PI-SceI and drop dialysis in nucleofection/electroporation/microinjection buffer is included in the purification service. Validation by additional restriction digestions (greater than 3) is available with commensurate fee schedule.  Purification is required to prevent excessive heat generation from arcing often due to high salt concentrations in DNA preps which can reduce the ES cell viability. 


technology
references
1: Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981 Jul 9;292(5819):154-6. PubMed PMID: 7242681.


2: Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci  U S A. 1981 Dec;78(12):7634-8. PubMed PMID: 6950406; PubMed Central PMCID: PMC349323.


3: Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 1987 Nov 6;51(3):503-12. PubMed PMID: 2822260.


4: Doetschman T, Gregg RG, Maeda N, Hooper ML, Melton DW, Thompson S, Smithies O. Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature. 1987 Dec 10-16;330(6148):576-8. PubMed PMID: 3683574.


5: Smithies O. Forty years with homologous recombination. Nat Med. 2001 Oct;7(10):1083-6. PubMed PMID: 11590419.

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