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knock-in (KI) - rat
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knock-in (KI) rat

The scarless targeted integration of functional DNA elements such as mutations, protein tags, fluorescent reporters and humanized genes into the genome for disease modeling as well as developmental and mechanistic studies is routinely achieved by RNA-guided nuclease technologies such as CRISPR-Cas. In addition to the ability to edit or delete DNA sequences within the genome, a repair process known as homology-directed repair (HDR) offers the ability to seamlessly insert DNA sequences.  In the presence of a double-stranded DNA break (DSB) created by the Cas endonuclease and an accompanying repair template (ssODN, lssDNA, donor vector) with sufficient homologous sequence, the cellular machinery may repair the DSB during the mitotic S-phase when homologous recombination proteins are active.  As a consequence, the repair template may become seamlessly integrated at the target site within the genome: this technology is known as a knockin (KI). 

Allow NovoHelix to leverage its expertise to build custom rodent models containing small knockins such as insertion of point mutations, altering amino acid codons, insertion of tags and barcodes, reporter genes (mKusabira-Orange, mCitrine, mVenus, mNeonGreen, GFP, mEmerald, mCherry, tdTomato, mScarlet, mCerulean, mTFP1, etc.) and whole gene replacement such as humanization. For large knockins, NovoHelix has routinely introduced large DNA constructs some upwards of 90-kb into traditional safe harbor sites Rosa26 and Aavs1 and has built a suite of flexible donor vectors such as Rosa26-lox-stop-lox (Rosa26-LSL). From the initial project concept, we can assist with a flexible design that includes building advanced genetic circuits for knockin to any genomic locus to advance your field.  
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Catalog Nr

Service Description

Timeline

Deliverables

Pricing

  • Custom knockin any locus
  • Rat Sprague Dawley KI via CRISPR microinjection 
  • guaranteed founders 
KIRN001
Services included are insertion of point mutations, altering amino acid codons, insertion of tags and barcodes, reporter gene knockins (GFP, mVenus, mCherry) and whole gene replacement such as humanization.

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

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

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  • Custom knockin any locus
  • Rat Sprague Dawley KI via CRISPR microinjection 
  • non-guaranteed service (per injection session) 
KIRN002

 Services included are insertion of point mutations, altering amino acid codons, insertion of tags and barcodes, reporter gene knockins (GFP, mVenus, mCherry) and whole gene replacement such as humanization.

 3 - 4 months founders 

 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.

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  • Custom knockin any locus 
  • custom rat strain - CRISPR-mediated knockin
  • guaranteed founders
KIRN003
 Services included are insertion of point mutations, altering amino acid codons, insertion of tags and barcodes, reporter gene knockins (GFP, mVenus, mCherry) and whole gene replacement such as humanization.

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

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

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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
 KIRN007

 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. Guide RNAs can also be tested in an vitro cutting assay if preferred, but we have found in vitro activity to supercede in vivo activity levels both in cellular and microinjection contexts; and hence in vitro mismatch nuclease assays often do not correlate to gene editing in vivo outcomes. 

 7-10 days

 Activity of up to 6 guide RNAs

  • Gene editing activity testing
  • Format - cell-based transfection
  • Assay - genetic reporter & flow cytometry
KIRN008
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

 Activity of up to 6 guide RNAs

 Genotyping Assay Development - knockin
KIRN009

 NovoHelix offers a knockout genotyping service to help clients develop robust genotyping protocols for screening animals after breeding and expansion of indidvidual mutant/KO founder lines.

 2-4 days

 genotyping protocol

 Genotyping Assay Development - complex knockin
KIRN010
 NovoHelix offers a knockout genotyping service to help clients develop robust genotyping protocols for screening animals after breeding and expansion of indidvidual mutant/KO founder lines.

 5-7 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)
KIRN011

 Service is for design and cloning of up to 6 guide RNAs for 1 target locust in a DNA plasmid format.

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)
 KIRN012
 Service is for design and in vitro transcription (IVT) 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)
KIRN013
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 
KIRN014
 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.
 Knockin dsDNA donor vector construction
KIRN015
 Isogenic knockin dsDNA donor vector construction and Sanger-sequence verified with up to 5-kb 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.
 Knockin dsDNA donor vector construction - complex
KIRN016
 Isogenic knockin dsDNA donor vector construction and Sanger-sequence verified with up to 50-kb 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
 KIRN017
 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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