conditional knockout (cKO)
In comparison to constitutive 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.
Service
Catalog Nr
Service Description
Timeline
Deliverables
Pricing
- flox allele / conditional knockout (cKO)
- C57BL/6 mouse via CRISPR microinjection
- guaranteed founders
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
- flox allele / conditional knockout (cKO)
- C57BL/6 mouse via CRISPR microinjection
- non-guaranteed service (per injection session)
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.
- flox allele / conditional knockout (cKO)
- custom mouse strain - CRISPR-mediated
- guaranteed founders
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
Service
Catalog Nr
Service Description
Timeline
Deliverables
Pricing
- Gene editing activity testing
- Format - cell-based transfection
- Assay - T7 endonuclease I/Cel-II/Surveyor
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
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
2-4 days
genotyping protocol
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
7-10 days
6 guide RNAs ~ 500 ng/ul --- RNA format