animal models / rat models /
conditional knockout (cKO) - rat
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conditional knockout (cKO) rat

In comparison to direct knockouts, conditional knockouts (cKO) allow for temporal and spatial control of gene disruption. To generate a cKO, the target gene’s critical exons are often flanked with directional loxP sites termed ‘floxed’ which can be excised by Cre recombinase. Crossing a Cre-expression driver mouse to this floxed allele affords the investigator to conditionally excise the targeted gene only in cells which express the recombinase Cre. Conditional gene disruption is useful for modeling embryonic lethal genes, tissue-specific gene deletion or developmental-stage gene disruption.  Our service packages include options such as guaranteed founders or pricing per microinjection session, flexible repair templates such as conditional donor vectors or long single-stranded DNA (lssDNA), and the choice of RNA molecule formats for guide RNA (gRNA) evaluation using the latest design guidelines:  full-length guides, synthetic single guide RNAs (sgRNAs), chemically-modified crRNA + tracrRNA or sgRNAs, truncated sgRNAs, & hairpin-sgRNAs (hp-sgRNAs). The advantage of testing multiple high-specificity Cas endonuclease variants and RNA molecule formats allows NovoHelix to tune the activity of custom RNP complexes to produce the highest on-target activity in our surrogate cell-based gene editing assays while limiting potential off-target activity. 
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model generation

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Service Description

Timeline

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Pricing

  • flox allele / conditional knockout (cKO)
  • Rat Sprague Dawley via CRISPR microinjection
  • guaranteed founders
CKOR001

 In comparison to direct knockouts, conditional knockouts (cKO) allow for temporal and spatial control of gene disruption. To generate a cKO, the target gene’s critical exons are often flanked with directional loxP sites termed ‘floxed’ which can be excised by Cre recombinase. Crossing a Cre-expression driver mouse to this floxed allele affords the investigator to conditionally excise the targeted gene only in cells which express the recombinase Cre. Conditional gene disruption is useful for modeling embryonic lethal genes, tissue-specific gene deletion or developmental-stage gene disruption.  The service includes donor vector or long single-stranded DNA (lssDNA) construction, guide RNA design using the latest design guidelines, i.e. full-length guides, chemically modified gRNAs, truncated sgRNAs, hp-sgRNAs (hairpin-sgRNAs), that produces high on-target activity in our surrogate cell-based gene editing assay while limiting off-target activity. 

 3 - 4 months founders; 5 - 8 months GLT F1s

 At least 2 cKO founders (or germline transmitted F1s) with the confirmed mutation

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  • flox allele / conditional knockout (cKO)
  • Rat Sprague Dawley via CRISPR microinjection
  • non-guaranteed service (per injection session) 
CKOR002

 Conditional knockout mice production with microinjection of the repair template/Cas RNP complex performed as a per session service with a total of 500 zygotes injected. The service includes donor vector or long single-stranded DNA (lssDNA) construction, guide RNA design using the latest design guidelines, i.e. full-length guides, chemically modified gRNAs, truncated sgRNAs, hp-sgRNAs (hairpin-sgRNAs), that produces high on-target activity in our surrogate cell-based gene editing assay while limiting off-target activity. 

 3 - 4 months founders; 5 - 8 months GLT F1s

 At least 500 embryos will be injected and implanted (2-3 microinjection sessions), which usually results in approximately 100 pups. Ear biopsies from all pups will be given to the investigator for genotyping. There is no guarantee that resulting pups will be gene edited and contain the floxed allele.

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  • flox allele / conditional knockout (cKO)
  • custom rat strain - CRISPR-mediated knockin
  • guaranteed founders
CKOR003

 Conditional knockout custom strain mice production via microinjection of the repair template/Cas RNP complex.  The service includes donor vector or long single-stranded DNA (lssDNA) construction, guide RNA design using the latest design guidelines, i.e. full-length guides, chemically modified gRNAs, truncated sgRNAs, hp-sgRNAs (hairpin-sgRNAs), that produces high on-target activity in our surrogate cell-based gene editing assay while limiting off-target activity. 

 3 - 4 months founders; 5 - 8 months GLT F1s

 At least 2 cKO founders (or germline transmitted F1s) with the confirmed mutation

Request Quote
support services

Service

Catalog Nr

Service Description

Timeline

Deliverables

Pricing

  • Gene editing activity testing
  • Format - cell-based transfection
  • Assay - T7 endonuclease I/Cel-II/Surveyor
CKOR004
NovoHelix offers a gene editing service to help clients test their CRISPR tools including guide RNAs, high-performance mutant Cas proteins and base editors in plasmid DNA or RNP formats.  A representative cell line will be transfected in triplicate and results will be generated by the mismatch-nucleases T7 Endo I or Cel-II as adopted from a protocol originally developed by Keith Joung's lab. While our cell-based assay is the gold standard for guide RNA activity, guide RNAs can be tested in an alternative in vitro cutting assay should the client prefer.  However, caution must be used in interpreting these in vitro results, as NovoHelix has broadly found that Cas9 RNP in vitro cutting of PCR amplicons as a surrogate assay for gRNA activity vastly overrepresents Cas RNP in vivo activity levels both in cellular and microinjection contexts. Hence in vitro mismatch nuclease assays often do not correlate to gene editing in vivo outcomes. In short, the cell-based gRNA testing is strongly suggested before proceeding to animal model generation.

 7-10 days

 Gene editing activity of up to 6 guide RNAs

  • Gene editing activity testing
  • Format - cell-based transfection
  • Assay - genetic reporter & flow cytometry
CKOR005

 NovoHelix offers a gene editing service to help clients test their CRISPR tools including guide RNAs, high-performance mutant Cas proteins and base editors in plasmid DNA or RNP formats.  A representative cell line will be transfected in triplicate and results will be generated by a fluorescent reporter assay and flow cytometry.

 7-10 days

 Gene editing activity of up to 6 guide RNAs

 Genotyping Assay Development - conditional knockouts
CKOR006
NovoHelix offers a knockout genotyping service to help clients develop robust genotyping protocols for screening animals after breeding and expansion of indidvidual mutant/cKO founder lines.

 2-4 days

 genotyping protocol

 Development of guide RNAs - guide RNA cloning and design in a mammalian (U6, H1) expression vector or T7 in vitro transcription (T7- IVT) vector (DNA format)
CKOR007

 Service is for design and cloning of up to 6 guide RNAs for 1 target locust.

 7-10 days

 6 guide RNAs in plasmid vectors

 Development of guide RNAs - guide RNA cloning and design with T7 in vitro transcription (T7- IVT) for use in RNP format (RNA format)
 CKOR008
 Service is for design and in vitro transcription of up to 6 guide RNAs for 1 target locust.

 7-10 days

 6 guide RNAs ~ 500 ng/ul --- RNA format

 Client-provided guide RNAs - preparation for microinjection (DNA/RNA format)
 CKOR009
Service is for purification and for preparation of gRNAs for zygotic microinjection or slide-electroporation. Client should provide validated gRNA synthesis data such as small RNA electrophoresis results from Agilent's Bioanalyzer prior to microinjection and a minimum 200 ng/ul gRNA concentration. Purification is required to prevent the microinjection glass needle from clogging, to prevent embryo lysis and extensive delays in refitting and resetting microinjection setup.
 Client-provided dsDNA donor vector - preparation for microinjection 
CKOR010
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 such as RNases and proteases.  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 compulsory to prevent the microinjection glass needle from clogging, to prevent embryo lysis and extensive delays in refitting and resetting the microinjection setup.
  •  cKO dsDNA donor vector construction
  • basic -  up to 5-kb floxed insertion
CKOR011
Isogenic cKO dsDNA donor vector construction and Sanger-sequence verified with up to 5-kb floxed insertion.  NovoHelix 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) chromotography 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.
  • cKO dsDNA donor vector construction
  • complex -  greater than 5-kb floxed insertion
CKOR012
 Isogenic cKO dsDNA donor vector construction and Sanger-sequence verified with up to 20-kb floxed insertion.  NovoHelix 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) chromotography 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.
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 Rat colony husbandry & management
CKOR013
 Rat colony management including breeding, weaning, sampling, monitoring, per cage per day for the production of founders and F-generation pups
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