The H9C2 line of embryonic rat
cardiomyocytes is a subclonal line of the original clonal cell line derived
from embryonic BD1X rat heart tissue by Kimes and Brandt (1976). This cell line
exhibits many of the properties of skeletal muscle and its parameters are
particularly useful in preclinical tests of anticancer drugs, determining their
cardiotoxicity, safety, and the possibility of moving to subsequent stages of
clinical tests. Therefore H9C2 cell line is commonly used in numerous in vitro
studies, some of which involve CRISPR technology to mediates the characteristics
of H9C2 cells.
H9C2 cells retain some components of the
signaling pathway essential for their differentiation into mature cardiac
muscle cells. The cell line is used, in particular, for cardiotoxicity analyses
of new, mainly anticancer drugs (e.g. doxorubicin), and studies on mechanisms
of myocyte damage, and assessment of toxic effects of studied compounds on
apoptosis and necrosis in cardiac myocytes. Embryonic H9C2 cardiomyocytes
proliferate well in vitro conditions, allowing relatively easy culturing and
are suitable to be a CRISPR gene knockout/knockin or over-expression model for
in vitro studies of cardiac hypertrophy and supports current work with human
cardiomyocyte cell lines for prospective molecular studies in heart development
and disease.
Applications:
1. Effects of C3G Knockout on Proliferation
and Apoptosis in H9C2 Cardiomyocytes
Previous studies found that C3G expression
was significantly increased in the myocardium of the non-infarct area around
the infarct in rats. Overexpressed C3G can promote cardiomyocyte survival and
inhibit cytotoxicity while knocking down C3G can inhibit cardiomyocyte survival
and increase cardiomyocyte apoptosis. The CRISPR/Cas9 system built-in knockout
C3G recombinant lentivirus was used to infect H9C2 cardiomyocytes to study the
speculated effect and mechanism of C3G knockout on the proliferation of H9C2
cardiomyocytes. H9C2 cardiomyocytes were infected with above lentiviruses
respectively to investigate the effects of C3G [Crk SH3-domain-binding guanine nucleotide
exchange factor] knockout on proliferation and apoptosis in H9C2 cardiomyocytes
and their underlying mechanisms.
2. Using CRISPR-Cas9 gene-editing
technology to knock out Tudor-SN gene of H9c2 cells to inhibit cell cycle
arrest and proliferation
CRISPR-Cas9 gene-editing technology was
used to knock out the Tudor-SN (Tudor staphylococcal nuclease) gene of rat
myocardial H9c2 cells. Researchers observed its effect on H9c2 cell cycle and
proliferation. The PX462 plasmid was selected as the vector, and the upstream
and downstream sgRNA (single-guided RNA) that can specifically recognize the
second exon of Tudor-SN gene in H9c2 cells were designed using software to
construct a pair of recombinant plasmids. Subsequently, the pair of plasmids
were co-transformed into H9c2 cells, and then positive monoclonal cells were
selected for cultivation. Western blotting was used to identify the knockout
effect, and the cell cycle and proliferation were detected by flow cytometry
and CCK-8=experiment using the successfully knocked out cell lines. Western
blotting results showed that Tudor-SN protein was not expressed in positive
cells, and the Tudor-SN gene was successfully knocked out. Flow cytometry
results showed that Tudor-SN gene knockout cells had a G1 phase arrest. The
results of the CCK-8 experiment showed that the proliferation rate of KO cells
slowed down. In this experiment, the Tudor-SN gene knockout cell line of H9c2
cells was successfully constructed, and the inhibition of Tudor-SN gene
knockout on cell cycle arrest and proliferation was detected, which provided a
study for the regulation of Tudor-SN gene on cardiomyocyte function. Convenient
tools and research foundation.
3. Dock180 knockout inhibits proliferation
and promotes apoptosis of rat derived H9C2 cardiomyocytes strain
To investigate the effects of dedicator of
cytokinesis 1 (Dock180) knockout on proliferation and apoptosis in rat derived
H9C2 cardiomyocytes and their mechanisms, a single guide RNA (sgRNA) targeting
rat Dock180 gene was designed and constructed using CRISPR/Cas9 system. A
plasmid contained above sgRNA was packaged into lentivirus and selected to
knockout Dock180 in the cardiomyocytes. The result showed that Dock180 knockout
with CRISPR/Cas9 H9C2 cells can inhibit proliferation and promote apoptosis via
p-ERK1/2, Bcl-2, and Bax in H9C2 cardiomyocytes.
Ubigene Biosciences, we provide high-efficiency and 100% guarantee CRISPR services including gene knockout/knock-in and point mutation cell lines. Make Genome Editing Easier is the goal of Ubigene. Our exclusive technologies have been successfully applied in more than 100 types of cell lines.
CRISPR-U ™ is an exclusive technology independently developed by Ubigene Bioscience for gene editing cell lines. By optimizing gene editing vectors and processes, the efficiency in gene-cutting and recombination of CRISPR-U ™ is 10 times higher than the conventional CRISPR / Cas9 technology. We are capable of customizing genetically-modified liver cell lines as you desire and satisfying various needs in gene editing.
Ubigene also provides CRISPR-B ™ gene-editing services exclusively
for bacteria and fungus, virus packaging such as lentiviruses, adenoviruses,
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References:
1. GAN Shi-Hu, CUI Xiao-Teng, MA Jin-Zheng,
FANG Li-Jiao, LIU Ming-Xia, REN Yuan-Yuan, CAO Xiao-Na, YANG Jie, SU Chao.
2.Using CRISPR-Cas9 gene-editing technology to knock out the Tudor-SN gene of
H9c2 cells to inhibit cell cycle arrest and proliferation. Chinese Journal of
Biochemistry and Molecular Biology, 2018.07.10.
2. Deng Qin, Liu Cheng, Zhang Jing, Li
Gang. The effect of knocking out C3G on the proliferation and apoptosis of H9C2
cardiomyocytes. Chinese Journal of Cell Biology: 1-6[2020-06-09].
3. HU Su-lei, LI Gang, FU Yan-bo, DENG Qin,
LIU Cheng. Dock180 knockout inhibits proliferation and promotes apoptosis of
rat derived H9C2 cardiomyocytes strain. Basic & Clinical Medicine. 2017,
37(4).
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