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Knockout and Knock-In Mouse







  • We can perform customers entire targeting project from initial strategy design or customer can outsource specific phases of the project to us
  • Our unique technology ensures a fast turn around time
  • Main strain use are C57BL/6 (B6) or FVB, but we can use any per your request
  • Our services include conventional knockout, conditional knockout and knock-in
  • If you wish to knockout your gene in the mouse quickly, you may consider nuclease-mediated knockout as an alternative

Diagram Knockout/Knock-In


THE SERVICE PROVIDED BY AMSBIO
SERVICES
DESCRIPTION
Transgenic Strategy Design
  • Analyze relevant information of your target gene, including gene structure, neighboring genes at the genomic locus, known targeted mouse models, etc.
  • Develop a gene targeting strategy, including the method for screening targeted ES cells by PCR and confirmation by Southern.
Transgenic Vector Construction
  • Clone relevant DNA fragments corresponding to homology arms into the targeting vector
  • Confirm the final targeting vector by restriction digest and sequencing
  • Types of Gene Targeting:
    1. Conventional Knockout
      • One or more critical exons of the target gene are replaced with a drug-selection cassette
      • This results in the permanent inactivation of the target gene in all cells of the body throughout development
    2. Conditional
      • One or more critical exons of the target gene are flanked by LoxP sequences which can be recognized by Cre recombinase
      • It is possible to specifically delete the floxed region and inactivate the gene in desired tissues, while in all other tissues, the target gene remains functional
    3. Conditional Knock-In
      • Reporter Knock-In
        • A visually detectable marker gene (such as GFP or lacZ) can be knocked into a gene locus to replace the coding sequence of the target gene for the purpose of monitoring the promoter activity of the gene
        • Alternatively, a marker or an epitope tag can be knocked into the end (or the beginning) of the coding sequence to form a fusion protein, which allows target gene expression and localization/trafficking to be examined
      • Point Mutation Knock-In
        • By introducing point mutation(s) into homology arms or the targeting vector, the desired mutation(s) can be incorporated into the target gene. The mutated gene is expressed under the control of wildtype gene regulatory elements
      • Humanized Mouse
        • This will be a mouse model in which a portion or the full mouse gene sequence has been replaced by its human counterpart. The human gene is expressed under the control of the mouse regulatory sequences
      • ROSA26 Knock-In
        • ROSA26 is a locus that has been widely used for achieving global expression of the introduced gene in mice. The gene of interest is targeted into the first intron of the locus. The expression of the gene can be driven by endogenous ROSA26 promoter or by a promoter of choice that is targeted into the locus along with the gene
Targeting in ES Cells
  • Electroporate linearized targeting vector into ES cells derived from B6, followed by appropriate drug selection, and pick drug-resistant clones
  • Screen for targeted ES cell clones by PCR.
  • Expand up to six PCR-positive clones, which are further confirmed by Southern
  • Optional: Remove drug-selection cassette in conditional knockout or knockin ES cells by FLP or Cre
  • Optional: Karyotype ES cells to ensure correct chromosome number
Chimera Production
  • Use either injection or aggregation to introduce targeted ES cells into host embryos followed by transfer into surrogate mothers
  • Score resulting pups for coat color chimerism
  • We guarantee delivery of >= 3 male chimeras wach with >50% chimerism
F1 Heterozygous Mutant Production
  • Breed male chimeras to B6 females
  • Genotype pups by PCR to identify F1 heterozygous mutants
  • We guarantee delivery of ≥2 germline F1 heterozygotes


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