animal models /
livestock models
/

livestock models


For decades, targeted genetic modification in large biomedical models had been notoriously tricky because homology-directed repair (HDR) occurs at a such a low frequency (often 1 in 10,000) in fibroblasts in comparison to gene targeting frequencies in traditional mouse ES cells (~1 in 100). With the advent of site-specific gene editing tools, chiefly CRISPR-Cas, a DNA double-stranded break (DSB) is intentionally introduced at the target site to stimulate homologous recombination (HR) with a frequency 50-1000-fold higher at the intended locus.  This increased HR activity results in a high rate of modification and, thus, reduces the number of fibroblast colonies to be screened by long-range PCR for the targeted allele.  The CRISPR-Cas technological platform accelerates the potential for seamless modifications by gene knockout and knockin. At NovoHelix, we have optimized gene editing techniques to address the critical need for large animal models across a variety of disciplines in biomedicine and translational research.  Please contact us through our online quoting system for project quotes and consultation. 
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model generation

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Catalog Nr

Service Description

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Pricing

Custom Genetic Modification -Livestock Biomedical Model
GELS001
NovoHelix scientists provide regular updates for livestock model generation milestones including isogenic targeting vector construction, establishment of primary cell lines from elite animals for somatic cell nuclear transfer (SCNT) and genome engineering, validation and expansion of single-cell gene-edited clones, transfer of SCNT embryos to recipient surrogates, pregnancies established and gestational outcomes, and delivery of the final gene-edited founder animals. 
12-24 months;   timelines vary depending on individual livestock gestational requirements.
 2 founders with the desired genetic modification
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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
 GELS004
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
GELS005

 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
 GELS006
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)
GELS007

 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)
GELS008
 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)
GELS009
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 
GELS010
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
GELS011
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) 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.
 cKO dsDNA donor vector construction - complex
GELS012
 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) 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.
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technology
references
1: Gao X, Nowak-Imialek M, Chen X, Chen D, Herrmann D, Ruan D, Chen ACH, Eckersley-Maslin MA, Ahmad S, Lee YL, Kobayashi T, Ryan D, Zhong J, Zhu J, Wu J, Lan G, Petkov S, Yang J, Antunes L, Campos LS, Fu B, Wang S, Yong Y, Wang X, Xue SG, Ge L, Liu Z, Huang Y, Nie T, Li P, Wu D, Pei D, Zhang Y, Lu L, Yang F, Kimber SJ, Reik W, Zou X, Shang Z, Lai L, Surani A, Tam PPL, Ahmed A, Yeung WSB, Teichmann SA, Niemann H, Liu P. Establishment of porcine and human expanded potential stem cells. Nat Cell Biol. 2019 Jun;21(6):687-699. doi:10.1038/s41556-019-0333-2. Epub 2019 Jun 3. PubMed PMID: 31160711.

2: Matoba S, Zhang Y. Somatic Cell Nuclear Transfer Reprogramming: Mechanisms and Applications. Cell Stem Cell. 2018 Oct 4;23(4):471-485. doi: 10.1016/j.stem.2018.06.018. Epub 2018 Jul 19. Review. PubMed PMID: 30033121PubMed Central PMCID: PMC6173619.

3: Estrada JL, Collins B, York A, Bischoff S, Sommer J, Tsai S, Petters RM, Piedrahita JA. Successful cloning of the Yucatan minipig using commercial/occidental breeds as oocyte donors and embryo recipients. Cloning Stem Cells. 2008 Jun;10(2):287-96. doi: 10.1089/clo.2008.0005. PubMed PMID: 18373474PubMed Central PMCID: PMC2981378.

4: Fischer K, Kraner-Scheiber S, Petersen B, Rieblinger B, Buermann A, Flisikowska T, Flisikowski K, Christan S, Edlinger M, Baars W, Kurome M, Zakhartchenko V, Kessler B, Plotzki E, Szczerbal I, Switonski M, Denner J, Wolf E, Schwinzer R, Niemann H, Kind A, Schnieke A. Efficient production of multi-modified pigs for xenotransplantation by 'combineering', gene stacking and gene editing. Sci Rep. 2016 Jun 29;6:29081. doi: 10.1038/srep29081. PubMed PMID: 27353424; PubMed Central PMCID: PMC4926246.

5: Niu D, Wei HJ, Lin L, George H, Wang T, Lee IH, Zhao HY, Wang Y, Kan Y, Shrock E, Lesha E, Wang G, Luo Y, Qing Y, Jiao D, Zhao H, Zhou X, Wang S, Wei H, Güell M, Church GM, Yang L. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science. 2017 Sep 22;357(6357):1303-1307. doi:10.1126/science.aan4187. Epub 2017 Aug 10. PubMed PMID: 28798043; PubMed Central PMCID: PMC5813284.
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