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Crispr KO of Cldn1 gene in mouse ES cells

Left panel: diagram illustrating the strategy for diagnosis of Crispr induced deletion. Right panel: gRNA sequence showing complementary binding to coding exon of Cldn1 gene and two positive ES cell clones carrying deletion in one allele of Cldn1 gene.

Crispr knockin of LoxP site into Cldn1 gene locus

Left panel: gRNA sequence as well as targeting vector sequence carrying LoxP site and a BamH1 site. Right panel: BamH1 digestion showing that ~5 ES cell clones out of 100 clones carry the LoxP site insertion into the cldn1 gene locus.

Deep sequencing results of double LoxP site insertion into Cldn1 gene locus

Out of 85 shortlisted ES cell clones carrying single LoxP site insertion at 5'-end or 3'-end of one allele of Cldn1 gene (yellow), 2 clones carry double LoxP site insertions into both alleles (green).

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Crispr Knockout & Knockin in ES Cells

$2,950.00 – $4,950.00

Item Cat No.: BCCRES

Application: Genome editing

Reactivity: Mouse ES cells

BiCell Scientific’s Crispr KO & KI service in mouse ES cells utilizes the Crispr-Cas9 system to edit a single allele in the mouse genome to achieve loss-of-gene function, gain-of-gain function, insertion of genetic materials and replacement with human DNA sequences.

CRISPR (clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotic organisms. Cas9 (or “CRISPR-associated protein 9”) is an enzyme that uses CRISPR sequences as a guide to recognize and open up specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes in mammalian cells.

CRISPR genome editing in mouse ES cells is the most efficient way to generate knockout and knockin mouse models. Because the edited allele can be segregated by mouse breeding, genome editing in mouse ES cells needs only to be done in one allele, which is less time consuming compared to genome editing in somatic cells.

BiCell Scientific’s Crispr KO & KI service in mouse ES cells guarantees success of genetic alteration as well as germline transmission after blastocyst injection. The genetically edited allele will be deep-sequenced to confirm the intended modification.

  • Molecular cloning of gRNA into pX330-U6-hSpCas9 vector.
  • Molecular cloning of targeting vector carrying intended mutation or insertion (KI projects only).
  • Electroporation of mouse ES cells of customer’s parental line with pX330-U6-hSpCas9 and targeting vector.
  • Perform Surveyor nuclease digestion (for KO projects) or qPCR (for KI projects) to identify positive clones.
  • Deep-sequencing positive clones to identify intended modification.

Customers will receive the following deliverables at the end of each project:

  • One frozen mouse ES cell clone with intended genetic modification (1 × 106 cells)
  • Deep-sequencing results

(Customers select the clone of their interest. BiCell Scientific Inc will bank the remaining clones. Additional clones are offered at $950 each).

For Research Use Only. Not for use in clinical diagnostics or therapeutics.

  • Molecular cloning of gRNA into pX330-U6-hSpCas9 vector and targeting vector – 2-3 weeks
  • Electroporation of mouse ES cells – 1-2 weeks
  • ES cells culture and clonal selection – 3-4 weeks
  • Surveyor nuclease test or qPCR test to shortlist positive clones – 2-3 weeks
  • Deep sequencing of shortlisted clones – 1-2 weeks

Additional information

Service type

KO, KI-point mutation, KI-reporter, Floxed-allele, Humanized-allele

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