Extreme Genome Editing (EGE) Technology
Strict Quality Control
Southern blot to screen out random insertions
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
Strict model validation protocols
Off-target effect prevention strategies
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.
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.
2) Flow analysis
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.