According to the characteristics of different cell lines, appropriate transfection methods (virus
transduction,
liposome transfection, and electroporation) will be selected to transfer gRNA sequences and Cas9
protein
into
cells, and antibiotic screening with different duration will be carried out according to different
transfection
methods used. After antibiotic screening, single-cell clones will be generated. Positive clones that
are
successfully knocked out will be validated by target site amplification and sequencing. Final
deliverables
will be
the homozygous KO cell clones, related data and project reports.
KO cell bank
3000 KO cell lines Starting from $1780 Deliver in 1 week!
Ubigene's KO cell bank covers thousands of genes, including 8 signal pathways, 10+ drug
development sectors, 100+ common diseases and popular research fields (such as m6A, ferroptosis
and exosomes). Search your target gene below to find out if there is a KO cell line in stock.
Knockout Cell Service
Cell type
Various types of cells including tumor cell lines, regular cell lines,
IPS/ES
cell lines
Human Breast Cancer Cell Line(MCF7)Mouse insulinoma β cell line(NIT-1)Human Breast Cancer Cell Line(JIMT-1)Human breast cancer cell line(T-47D)Human pancreatic cancer cell line(BxPC-3)Mouse Acinar Pancreatic Cell Line(266-6)Human Prostate Cancer Cell Line(VCaP)Human Pancreatic Carcinoma Cell Line(MIA PaCa-2)Mouse medullary breast cancer cell line(E0771)Mouse pancreatic cancer cell line(Pan02)Human Metastatic Pancreatic Adenocarcinoma Cell Line(AsPC-1)Human Breast Adenocarcinoma Cell Line(SK-BR-3)Human Pancreatic Carcinoma Cell Line(PANC-1)Rat Breast Cancer Cell Line(4T1)Human Breast Cancer Cell Line(ZR-75-1)Human Breast Cancer Cell Line(MDA-MB-231)
Guide RNAs target introns at both sides of exon 2 and the number of bases in exon 2 is not a
multiple
of 3,
which can cause frame-shift mutation.
Frame-shift
mutation
Guide RNA targets the exon, and the base number of deletion is not a multiple of 3. After
knockout,
frame-shift mutation would cause gene knockout.
Large
fragment
removal
Complete removal of the coding sequence to achieve gene knockout.
Work Flow and Validation
Case Study
Chinese hamster ovary (CHO) cells have been used as host cells in the production
of a
range of recombinant therapeutic proteins,
including
monoclonal
antibodies and Fc-fusion proteins. Host cell proteins (HCP) represent impurities that must be
removed from
therapeutic formulations because of their potential risks for immunogenicity. While the majority of
HCP
impurities
are effectively removed in typical downstream purification processes, clearance of a small
population of HCP
remains challenging. Using the CRISPR/Cas9 system, Anxa2-, and Ctsd-knockout CHO cell lines were
successfully
established, and this study confirmed the complete elimination of the corresponding HCP in cell
lysates.
Importantly, all knockout cell lines showed similar growth and viability to those of the wild-type
control
during
8 days of cultivation. Thus, knockout of unrequired genes can reduce contamination with HCP in the
production of recombinant therapeutic proteins.
（a）The constructed sgRNA targeted a unique sequence in exon 4 of the Anxa2 gene.
The
amplicon sequence was analyzed in both directions.
（b）The constructed sgRNA targeted the unique sequence in exon 2 of the Ctsd
gene.The
amplicon sequence was analyzed in both directions.
Characterization of protein expression from CHO knockout cell lines by SDS-PAGE
and
western
blotting analysis. (a) Anxa2-knockout cell lines. (b) Ctsd-knockout cell lines. Cell
culture
supernatants and cell lysates were subjected to SDS-PAGE. Total proteins were detected by CBB
staining.
Western
blotting analysis of each protein was performed using respective capture and detection antibodies.
The
asterisk indicates the nonspecific band. The double asterisk indicates what is likely a fragment of
cathepsin D.
Annexin A2 and cathepsin D were not detected in the cell culture supernatants or
lysates
of
the corresponding knockout cell lines, suggesting successful exclusion of these impurities from host
cells
for
therapeutic protein production. In addition, no truncated HCP was observed in the protein expression
analysis of
the Anxa2 and Ctsd knockout cell lines.
Reference：
Fukuda, N., Senga, Y., & Honda, S. (2019). Anxa2‐and Ctsd‐knockout CHO cell
lines to
diminish the risk of contamination with host cell proteins. Biotechnology progress, e2820.
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