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Production of Conditional Knockout Mice

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Production of Conditional Knockout Mice

Production of Conditional Knockout Mice

The conditional knockout method is also known as conditional gene disruption. The targeted gene region is sandwiched by loxP sequences recognized by Cre recombinase. By crossing a mouse with this allele, called flox mouse, with a specific Cre-expressing mouse, it is possible to delete the targeted gene only in cells which express Cre. It is also possible to disrupt gene in stage specific manner by using inducible Cre recombines (e.g. CreER). Recently, conditional knock out is extremely useful methods to analyzefunctions of various genes.

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Features

・Fatal genes can be analyzed.
・Organ-specific genes can be disrupted.
・Genes specific to certain stages of development can be disrupted.
・Mice are also created with vectors from KOMP and EUCOMM.
・We have just started “Skip Service”, in which chimera mice are directly crossed with CAG-Flp mice to obtain flox mice in shorter period.

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Outline of Production Work

Process Contents Standard time period Price
Construction of targeting vector 1. Collection of the information about target gene from database, and design of targeting strategy 2~3 months JPN1,100,000
2. Cloning of genomic sequences for homologous recombination
3. Construction of targeting vector by subcloning genomic sequences and drug resistant cassettes with loxP and FRT
4. Construction of control vector to set up screening homologous recombinants
Establish homologous recombinant ES cell clones 5. Electroporation of targeting vector into ES cells 3~4 months JPY1,800,000
6. Isolation of drug resistant ES cell clones by picking up colonies
7. Screening homologous recombinant by PCR
8. Establishing homologous recombinant ES clones by PCR analysis and Southern blot analysis with neo probe.
Production of chimeric mice 9. Production of chimeric mice with max. 3 clones of homologous recombinant ES cell clones (If contribution of ES cells is less than 80% in all chimeric mice, chimeric mice are re-produced). Approx. 3 months JPY700,000
Production of heterozygous mice 10. Identification of germline-transmitted chimeric mice by natural mating (mating 4 chimeric mice with highest contribution of ES cells with wild-type mice). Approx. 3 months JPY700,000
11. Production of F1 heterozygous mice
Total *Health status report and transportation fee for delivery are not included JPY4,300,000
Elimination of drug resistant gene (production of flox heterozygous mice) Optional, F1 heterozygous mice are crossed with CAG-Flp mice (or CAG-Cre mice) to elminate drug resistant gene. Then next generation is crossed with wild-type mice with wild-type mice to remove Flp (or Cre) transgene. Approx. 6 months JPY650,000

* We also offer mating of chimeric mice with CAG-Flp mice to obtain germline transmitted mice and eliminate drug resistant gene in same time to save time.

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Details of Work

STEP 1: Design of Vector for Use in Genetic Modification

First, the database is searched for your gene of interest. The appropriate exon(s) for complete disruption of the anticipated protein function is selected. We can also design various strategies to modify genetic composition according to your request. Then, we propose strategy of gene targeting by homologous recombination.

STEP 2: Formal Contact

STEP 2: Formal Contact

Through this contract, a formal arrangement is established. A confidentiality agreement is also concluded at the same time as the formal contract.

STEP 3: Construction of Vector for Use in Genetic Modification

We provide a more detailed proposal for the vector, which we then begin construction. Genomic sequences for homologous recombination (homology arms) are amplified by PCR and cloned.

STEP 4: Establishment of Recombined ES Cells from Gene Introduction

The constructed targeting vector is introduced into the ES cells by electroporation. Homologous recombinant ES cell clones are identified by 1st screening PCR, detailed analysis by PCR, and Southern blot analysis with neo probe, and established.

STEP 4: Establishment of Recombined ES Cells from Gene Introduction

STEP 5: Production of Mouse from Established Recombined ES Cells

Mice are produced from the now-established homologous recombinant ES cells. A chimeric mouse is produced from maximum 3 clones of the established ES cells.

STEP 5: Production of Mouse from Established Recombined ES Cells

STEP 6: Delivery

The generated live mice are delivered from our company at the convenience of the customer. We can deliver F1 heterozyous mice, flox mice, chimeric mice. We also offer production of homozygous mice, mass production of flox mice and many other options. Please inquire us.

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