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Production of Conventional Knockout Mice

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Production of Conventional Knockout Mice

Production of Conventional Knockout Mice

Trans Genic Inc. offers the production service of conventional knockout mouse (also known as null knockout orgene disruption). Important exon(s) of the targeted gene is replaced with a drug-resistant gene to disrupt product function in all the cells of mouse body.
Please let us know your gene of interest. We present the design of targeting vector or your gene based on the available genetic information.

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Outline of Production Work

  Process Contents Period Price
01. Construction of targeting vector 1. Collection of the information about target gene and design of targeting strategy Approx. 2 months JPY800,000
2. Cloning of genomic sequences for homologous recombination
3. Construction of targeting vector by sucloning genomic sequences and drug resistant cassettes
4. Construction of control vector to set up screening homologous recombinants
02. Establish homologous recombinant ES cell clones 5. Electroporation of targeting vector into ES cells Approx. 3 months JPY1.400,000
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.
03. 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
04. 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
05. Delivery Health status report According to the regulation of your facility, health status report is prepared. Approx. 1 month Additional estimation
Delivery Transportation of mice
Total
(Except health status report, transportation fee and other options)
JPY3,600,000
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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 interst. the gene desired by the customer. The appropriate exon(s) for complete disruption of the anticipated protein function is selected. 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

After confirming that thehomologous recombinant ES cells have been transmitted to the germline, heterozygous mice are produced.

STEP 6: Delivery

The mice are delivered from our company at the convenience of the customer. Normally two pairs of heterozygous mice are delivered, but we can also deliver chimeric mice or honozygous mice. Prior to delivery we set up PCR condition for genotyping.

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