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Knockout Cell Line

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 $1980

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
Service type	Single / Multiple Genes Knockout
Deliverables	Cell pool/Single-cell clone
Turnaround/Price	Speedy turnaround as fast as 4 weeks! Contact Us

Urinary System

Human bladder carcinoma cell line (TCCSUP)
Human bladder carcinoma cell line (5637)
Human Bladder Transitional Cell Carcinoma Cell Line (T24)
Human Embryonic Kidney Cell Line(HEK293)
Human Embryonic Kidney Cell Line(293T)
Human prostate carcinoma cell line (22RV1)
Madin-Darby Canine Kidney Cell Line (MDCK)
Distal nephron cell line (Distal nephron cell line（JU4s）)

Blood and Lymphatic System

Mouse Macrophage Cell Line (RAW264.7)
Porcine Alveolar Macrophage Cell Line (3D4/21)
Human Monocytic Cell Line (THP-1)
Rat Basophil Leukemia Cell Line (RBL-2H3)
Human leukemia cell line (HL-60)
Human T lymphocyte cell line (Jurkat, Clone E6-1)
Human myelogenous leukemia cell line (K-562)
Human caucasian histiocytic lymphoma cell line (U-937)
Human Acute Non-B Non-T Lymphocytic Leukemia Cell Line (Reh)
Human B Cell Lymphoma Cancer Cell Line U2932

Respiratory System

Human Lung Cancer Cell Line (A549)
Human Lung Cancer Cell Line (Calu-1)
Human Lung Squamous Carcinoma Cell Line (NCI-H520)
Lewis lung carcinoma (LLC)
Human Lung Cancer Cell Line (SK-MES-1)
Human Lung Squamous Cell Carcinoma Cell Line NCI-H226
Human Non-small Cell Lung Carcinoma Cell Line (NCI-H1299)
Human Non-small Cell Lung Carcinoma Cell Line (HCC827)
Human Small Cell Lung Cancer Cell Line H69AR
Human Bronchial Epithelial Cell Line (BEAS-2B)
Human Bronchial Epithelial Cell Line (16HBE)
Human hypopharyngeal carcinoma cell line (FaDu)
Chinese hamster lung cells (V79)

Endocrine System

Human Breast Cancer Cell Line (MDA-MB-231)
Human Breast Cancer Cell Line (MDA-MB-468)
Human Breast Cancer Cell Line (MDA-MB-453)
Human Breast Cancer Cell Line (MDA-MB-436)
Human Breast Cancer Cell Line (ZR-75-1)
Human Breast Epithelial Cell Line (HBL-100)
Human Breast Cancer Cell Line (MCF7)
Mouse Breast Cancer Cell Line (4T1)
Human Pancreatic Carcinoma Cell Line (PANC-1)
Human Breast Cancer Cell Line (JIMT-1)
Human breast cancer cell line (MCF-7)
Human Breast Adenocarcinoma Cell Line (SK-BR-3)
Human Metastatic Pancreatic Adenocarcinoma Cell Line (AsPC-1)
Mouse Pancreatic Cancer Cell Line (Pan02)
Murine Breast Cancer Cell Line (E0771)
Human Pancreatic Carcinoma Cell Line (MIA PaCa-2)
Human prostate cancer cell line (PC3)
Human Prostate Cancer Cell Line (VCaP)
Mouse Acinar Pancreatic Cell Line (266-6)
Human pancreatic cancer cell line (BxPC-3)
Human Breast Adenocarcinoma Cell Line (T-47D)

Circulatory System

Rat Cardiac Myocytes (H9C2)
Mouse Cardiac Muscle Cell Line (HL-1)
Human Coronary Artery Endothelial Cell line (HCAEC)
Mouse Myoblast Cell Line (C2C12)

Digestive System

Human Colon Cancer Cell Line (HCT116)
Human Colon Cancer Cell Line (SW480)
Human Colon Cancer Cell Line (SW620)
Human Colon Cancer Cell Line (HT-29)
Human Colon Cancer Cell Line (LoVo)
Human colon carcinoma cell line (RKO)
Human caucasian colon adenocarcinoma cell line (COLO 205)
Murine Colorectal Carcinoma Cell Line (MC38)
Murine colorectal carcinoma cell line (CT26.WT)
Human colon adenocarcinoma cell line (DLD-1)
Human colorectal adenocarcinoma cell line (NCI-H716)
Human colorectal adenocarcinoma cell line (Caco-2)
Human colon carcinoma cell line (T84)
Human Liver Cell Line (L-02)
Human liver cancer cell line (Hep G2)
Human Hepatoma Cell Line (Hep 3B)
Human hepatocellular carcinoma cell line (HuH-7)
Human Hepatocellular Carcinoma Cell Line (SNU-387)
Human Hepatocarcinoma Cell Line (SMMC-7721)
Mouse Hepatoma Cell Line (H22)
Mouse Hepatoma Cell Line (Hepa 1-6)
Human hepatobiliary cancer cell line (RBE)
Human Gastric Cancer Cell Line (HGC-27)
Human Gastric Cancer Cell Line (SGC-7901)
Human Gastric Cancer Cell Line (MGC80-3)
Human gastric cancer cell line (AGS)
Human Esophageal Squamous Carcinoma Cell Line (KYSE-150)
Human Esophageal Squamous Carcinoma Cell Line (KYSE-30)
Human renal cell carcinoma cell line (786-o)
African green monkey kidney cell (Vero)
Human Renal Cell Carcinoma Cell Line (ACHN)

Skeleton, Articulus, Soft Tissue, Derma System

Human Osteosarcoma Cell Line (MG63)
Human bone osteosarcoma epithelial cell line (U-2 OS)
Human fibrosarcoma cell line (HT1080)
Human malignant melanoma cell line (A-375)
Human Melanoma Cell Line (M14)
Murine melanoma cell line (B16-F10)
Human Epidermoid Carcinoma Cell Line (A431)
Mouse myeloma cell line (Sp2/0-Ag14)
Mouse Skin Cancer Cell Line (SCC7)
Immortalized Ameloblastoma Cells (hTERT-AM)

Ocular, Otolaryngologic and Oral System

Rat Muller Cell Line (rmc-1)
Human Nasopharyngeal Carcinoma Cell Line (NPC-43)
Human Nasopharyngeal Carcinoma Cell Line (cne2z)
Human Nasopharyngeal Carcinoma Cell Line (CNE-1)

Brain and Nervous System

Human Neuroblastoma Cell Line (SK-N-SH)
Human Neuroblastoma Cell Line (SH-SY5Y)
Mouse neuroblastoma cell line (Neuro-2a)
Human Glioblastoma Cell Line (U251)
Rat Glioblastoma Cell Line (C6)
Mouse Glioblastoma Cell Line (GL261)
Mouse Microglial Cell Line (BV2)
Human glioblastoma cell line (U-87 MG)
Mouse Anterior Parietal Bone Cell Line (MC3T3-E1 Subclone 14)
Human Microvascular Endothelial Cell Line (hCMECD3)
Immortalize Human Microvascular Endothelial Cell Line (hCMEC/D3)
Mouse Hippocampal Neuron Cell Line (HT22)

Reproductive System

Human Cervical Carcinoma Cell Line (HeLa)
Human Cervical Carcinoma Cell Line (Hela 229)
Human Cervical Squamous Cell Line (SiHa)
Mouse Embryonic Fibroblasts (NIH/3T3)
Chinese Hamster Ovary Cell Line (CHO-K1)
Human Ovarian Cancer Cell Line (SK-OV-3)
Human Ovarian Cancer Cell Line (OVCAR3)
Human Choriocarcinoma Cell Line (BeWo)
Human Ovarian Cancer Cell Line (CAOV3)
Human Trophoblast Cell Line (HTR-8/SVneo)
Mouse Pituitary Gonadotrope Cell Line (Lbetat2)
Mouse Testicular Stromal Cell Line (TM3)

CRISPR-U™ Technical advantage

Higher cutting and homologous recombination efficiency, 10-fold increase in gene-editing efficiency

A large number of success case, successfully achieving over 5000 gene edits in over 200 cell line types

Wide application, easily achieving gene knockout / point mutation / knock-in

Knockout Strategies

Short fragment removal

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.

Anxa2 gene knockout cell line, construction of Ctsd gene knockout cell line

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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