53 Life Technologies
™
| Genome editing
Application example: Improve genome editing outcomes in biologically-relevant cell models.
Read the full application note here.
Ordering information
Product Quantity Cat. No.
GeneArt
®
Genomic Cleavage Detection Kit 20 reactions A24372
Introduction
With increasing expansion into research areas
of more biological relevance, existing molecular
and cellular techniques need to be improved.
Lipofectamine
®
3000, a new transfection reagent
developed to improve delivery and enable use of new
technologies, can be used in more relevant systems
enabling faster and more reliable outcomes. The area
of genome editing is rapidly growing and requires
more advanced techniques to maximize its potential
applications. Transcriptional activator-like effector
nucleases (TALENs) and technology derived from
clustered regularly interspaced short palindromic
repeats (CRISPRs) allow precise cleavage of DNA at
specifi c loci. However, the effectiveness of these tools
is contingent upon the intrinsic properties of the locus
of interest, effi cient delivery, and the painstaking
downstream processes of generating stable cell
lines and knockout models to study the phenotypic
effects of such genetic modifi cations. During the
development of Lipofectamine
®
3000 Reagent, we
assessed transfection of HepG2 and U2OS cell lines
with TALEN and CRISPR vectors designed using
GeneArt
®
Gene Synthesis services to target a specifi c
locus. We observed improvements in transfection
effi ciency, mean fl uorescence intensity, and genomic
cleavage. These advancements in delivery help
minimize painstaking downstream workfl ows,
enable easier stem cell manipulation, and enhance
site-specifi c insertion of transgenes into the cellular
genome.
Materials and methods
Plasmid design and preparation
GeneArt
®
Precision TALs and GeneArt
®
CRISPR
Nuclease Vectors were designed using the Life
Technologies GeneArt
®
web design tool
(lifetechnologies.com/us/en/home/life-science/
cloning/gene-synthesis/geneart-precision-tals.html).
The forward and reverse TALENs contain the FokI
nuclease and target the AAVS1 safe harbor locus.
The all-in-one CRISPR vector system contains a
Cas9 nuclease expression cassette and a guide
RNA cloning cassette that target the AAVS1 safe
harbor locus, combined with a downstream orange
fl uorescent protein (OFP) reporter. A negative control
plasmid, pcDNA
™
3.3, was also used throughout
the assay. The plasmids were transformed into
competent E. coli cells. Clones were analyzed and
sequenced for specifi city and then purifi ed using
a PureLink
®
HiPure Plasmid Filter Maxiprep Kit to
ensure low endotoxin activity and high-quality DNA.
Improve genome editing outcomes in
biologically relevant cell models
APPLICATION NOTE Lipofectamine
®
3000
