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Custom Production of Genetically Engineered Mice

Production of Knock-in Mice

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  • 2013/05/27
    TRANS GENIC launched contract knock-in mouse production service utilizing Rosa26 gene

We have started Rosa26 knock-in service in discounted price.
Rosa26 refers to a locus that is widely used for achieving generalized expression in the mouse, initially identified by retroviral gene trap vector (Friedrich and Soliano, 1991).

Rosa26 locus has following useful features;
・Endogenous promoter activity for ubiquitous expression.
・Minimum influence by epigenetic repression.
・High efficiency for homologous recombination.
・No phenotype in homozygous knockout mice.
These features make it possible to establish superior knock-in (KI) mouse line expressing your gene of interest (GOI).

Trans Genic Inc. offers a versatile vector system to produce various Rosa26 KI mouse lines. Followings are examples of our Rosa26 KI vectors (please see attached file for the detail);
1, Expression from Rosa26 promoter ; your GOI is ubiquitously expressed.
2, Expression from exogenous promoter ; expression of your GOI is regulated by any promoters of your choice.
3, Inducible expression from Rosa26 promoter ; expression of your GOI is induced in tissue specific or time specific manner in combination with various Cre mouse lines.

Overall, Rosa26 KI system provides stable and efficient expression of your GOI, which is superior to conventional transgenic mouse system.

Production of Knock-in Mice

Production of Knock-in Mice

The knock-in mouse is a method in which a exogenous gene is introduced into the mouse genome to analyze product function and/or expression pattern. Exogenous gene can be introduced to disrupt the endogenous gene, generating the knockout/knock-in mouse.. It is also possible to generate inducible gene expression strategy with Cre/loxP system, ubiquitous gene expression strategy using ROSA26 locus, and other various strategies. Combining with a reporter protein (LacZ, GFP, etc.) to investigate the expression pattern of mouse gene is also included inthe production of knock-in mice. In the custom production of genetically engineered mice conducted by our company, the production of knock-in mice is what requires the most advanced technology.
We propose strategies to make various gene engineered mice according to your requirement.

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Outline of Production Work

Process Contents 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

(JPY300,000 for ROSA26-KI)
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

(JPY1,000,000 for ROSA26-KI)
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

(JPY2,700,000 for ROSA26-KI)
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 we investigate about your exogenous gene to be knocked-in. If you do not have the source of knock-in gene, we propose a plan and an estimate for synthesizing the full-length DNA (additional charge required). We also provide a proposal for cloning the region around the knock-in gene. We recommend to create a design in which the drug-resistant genes are removanle by recombinase in the future to eliminate the possibility of interference.

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.
Cloning the knock-in gene is also necessary, so knock-in mouse production requires longer time than the production of other genetically engineered mice.

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 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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