Biocytogen 

Gene Targeting Services

>CRISPR/EGE Based Gene KO/KI Targeting



Increase homologous recombination efficiency by 10-20 fold 

when compared to standard CRISPR-Cas9!


Extreme Genome Editing (EGE) Technology


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Service Workflow 


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Strict Quality Control


Southern blot to screen out random insertions 


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Statistical analysis showed that for the ESC/HR method , the random insertion rate is approximately 20% and for EGE® technology, the rate is about 32%. Out of that 32%, 14% of the insertions cannot be removed through mating and passaging. Southern blot analysis is the gold standard to test for random insertions. Biocytogen recommends Southern blot analysis to ensure that there are no random insertions in our gene edited animal models.


Strict model validation protocols


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Off-target effect prevention strategies


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Application Examples 


1. Knockin rat


In February 2014, Biocytogen successfully developed a CD4-dsRed and FoxP3-DTR-EGFPreporter gene knockin rat using EGE technology. As of January 2017, Biocytogen has generated more than 2,000 knockin rat/mouse models for customers using EGE , making Biocytogen a leader for the development of gene modified animal models.


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As shown above, a pCAG-loxP-3 X STOP-loxP-tdTomato-WPRE-bGHpA cassette is inserted between the first and second exons of the Rosa26 site with EGEⓇ technology. After mating cKI rats with Crh-Crerats, tdTomato is highly expressed in the hippocampus of the Crh-Cre/tdTomato offspring.


2. Gene knockin cell line


A knockin cell line model was generated with EGFP inserted at the translation initiation site of ACTB (cytoskeleton filiform protein) in the U2OS cell line (human osteosarcoma). Another cell line model is EGFP-LMNB1 (nuclear envelope protein) knockin in C6 cells (rat brain glioma).


1) Targeting vector design and fluorescence detection result


ACTB gene encodes β-ACTIN protein which is a cytoskeletalprotein. The homologous arm of the targeting vector is about 1kb, and the homologous recombination of target vector is achieved by using EGE technology.

LMNB1 encodes the nucleoprotein Lamin-B1. The homologous arm of targeting vector is about 1kb, and the homologous recombination is achieved by using EGE  technology.


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2) Flow analysis 


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In U2OS cells, flow cytometryplots show that the efficiency of EGFP-ACTB gene knockin mediated by EGE(15.02%) is 8 times higher compared to CRISPR/Cas9-mediated knockin(1.91%). In the C6 cell line, the EGE system improves the knockinefficiency of EGFP-LMNB1to 3.6% from 0.19%, which is a 19-fold increase.


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