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Gene Targeting Technology Based on Embryonic Stem Cell (ESC/HR)

Gene targeting in embryonic stem cells with homologous recombination is a technology used to integrate an exogenous gene site-specifically into a certain locus in the target cell genome via homologous recombination. This method can modify a specific locus and engineer a gene onto a certain chromosome. Gene targeting technology has been widely regarded as the best method to specifically modify and engineer the genetic material of living body. The establishment of a conditional and induced gene targeting system makes the target position modification to the gene more definite in terms of time and space, and makes the gene's effect more accurate and reliable. This technology's development has provided a new method to study developmental biology, molecular genetics, immunology and medical science, and has large application prospects and prospective business value.

1. Model preparation flow       

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2. Customized gene targeting mouse model service flow

BIOCYTOGEN provides a one-stop, customized mouse model construction service, including model design, construction, and genotyping identification service.

2.1 Project feasibility evaluation and model design

BIOCYTOGEN possesses a senior technical team specializing in comprehensive analysis of project feasibility, and provides a free and professional model design service. The services include:

Ÿ Define the purpose for model construction

Ÿ Analyze the structure and sequence information of the target gene

Ÿ Analyze  existing data

Ÿ Design all possible model construction schemes

Ÿ Analyze all model construction schemes’ advantages and risks

Ÿ Accurate analysis of project implementation time and project quotation

2.2 Design and construction of the targeting vector

Ÿ Design the construction strategy of the targeting vector

Ÿ Design the positive clones screening strategy via PCR and Southern blot

Ÿ Subscribe the BAC

Ÿ Construct the targeting vector

Ÿ Sequencing of targeting vector

Ÿ Targeting vector linearization for the ES cell electroporation

2.3 ES cell electroporation and drug screening

Ÿ Recovery and amplification of ES cell

Ÿ Electroporation of ES cell with targeting vector

Ÿ Drug screen ES cell

Ÿ Select 200 to 400 cell clones at every electroporation time

Ÿ Cell clones amplification

Ÿ DNA extraction of cell clones to screen positive ES cell clones

2.4 Screening of positive ES cell clones

Ÿ Screen positive ES cell clones via short fragment PCR, long fragment PCR, and Southern blot, and ensure there is no random insertion

Ÿ Guarantee the ES cell heredity stability as well as efficiency of phyla heredity with karyotype analysis

Ÿ ES cell amplification for use in microinjection

2.5 Microinjection

Ÿ Prepare embryo donor mice and surrogate mice

Ÿ Microinject the ES cell clone into the blastocyst for blastocyst transfer

2.6 Procurement of chimeric mice and breeding of chimeric mice

Ÿ Feeding of recipient female mice and chimeric mice birth

Ÿ Mating of chimeric mice and wild mice

2.7 Obtain the phyla-genetic hybrid mice of generation F1

Ÿ Birth of generation F1 mice

Ÿ Ensure the phyla heredity by identification of generation F1 mouse tail genotype

3. Gene knockout mouse model

Gene knockout model: the model lacks the target gene or the target gene is inactivated. The loss of gene activity often causes a change in the animal model's phenotype, and the model can thus be used in in vivo analysis of gene function, disease studies, and drug development.


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