knockin cell line

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Location: Home > Gene Editing Services > Stable Cell Lines > knockin cell line

Knockin Cell Line

The gRNA vector (containing Cas9) of the CRISPR/Cas9 system and the Donor vector carrying the target mutation or target gene sequences will be co-transfected into cells via electroporation. Subsequently, the gRNA-Cas9 complex will induce a double-strand break (DSB) at the target site on the DNA. The cells will then undergo homologous recombination repair (HDR), using the Donor vector as a template to integrate the target mutations into the target sites. After antibiotic selection, single-cell clones will be generated. Positive clones that have successfully incorporated the target mutations will be validated through amplification and sequencing of the target sites. The final deliverables will include the validated positive cell clones, related experimental data, and project reports.

Understanding Common Methods for Point Mutation Research>>

EZ-HRex™ New Technique
After years of R&D of Ubigene, based on the original CRISPR-U™ gene-editing technique, Ubigene has upgraded it to EZ-HRex™ New Technique, by innovatively adding U+ Molecule.
Effectively regular cell cycles, promoting more post-transfection cells into S/G2 phases.
Reduce the NHEJ pathway activity, reducing the proportion of indel genotype to improve the HDR efficiency.
By reducing the activity of the NHEJ pathway, the proportion of indel genotypes is decreased, thereby enhancing HDR efficiency. Following optimization, the HDR efficiency for gene mutation and fragment knock-in in cells has been significantly improved across the board. At the post-transfection cell pool level, the proportion of HDR genotypes can reach up to 84%.

Knockin Cell Service Details

Cell typeVarious types of cells including tumor cell lines, regular cell lines, IPS/ES cell lines
Service typeRNP Method, Prime Editor, Base Editing, Antibiotics-based Konck in
DeliverablesSingle-cell clone
Turnaround/Price8 weeks,as low as $6480   Contact Us

300+Successful
Gene-editing Cell Line Types

Respiratory System
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Chinese hamster lung cells(V79)Human hypopharyngeal carcinoma cell line(FaDu)Human Bronchial Epithelial Cell Line(16HBE)Human Bronchial Epithelial Cell Line(BEAS-2B)Human Non-small Cell Lung Carcinoma Cell Line(HCC827)Human Non-small Cell Lung Carcinoma Cell Line(NCI-H1299)Human Lung Squamous Cell Carcinoma Cell Line(NCI-H226)Human lung squamous cell carcinoma cell line(SK-MES-1)Human Lung Cancer Cell Line(NCI-H520)Human Lung Cancer Cell Line(Calu-1)Human Lung Cancer Cell Line(A549)
Circulatory System
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Mouse Myoblast Cell Line(C2C12)Human Coronary Artery Endothelial Cell line(HCAEC)Rat Cardiac Myocytes(HL-1)Rat Cardiac Myocytes(H9C2)
Endocrine System
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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)
Brain and Nervous System
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Human glioma cell line(U251MG)Mouse microglia cell line(BV2)Immortalize Human Microvascular Endothelial Cell Line(hCMEC/D3)Mouse Anterior Parietal Bone Cell Line(MC3T3-E1 Subclone 14)Human glioblastoma cell line(U-87 MG)Rat Glioblastoma Cell Line(C6)Mouse neuroblastoma cell line(Neuro-2a)Human Neuroblastoma Cell Line(SK-N-SH)
Blood and lymphatic System
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Human B lymphoma cell line(Su-DHL-4)Human B Cell Lymphoma Cancer Cell Line OCI-LY3(OCI-LY3)Human B Cell Lymphoma Cancer Cell Line(U2932)Human Acute Non-B Non-T Lymphocytic Leukemia Cell Line(Reh)Human myelogenous leukemia cell line(K-562)Human T lymphocyte cell line(Jurkat, Clone E6-1)Rat Basophil Leukemia Cell Line(RBL-2H3)Human Monocytic Cell Line(THP-1)Porcine alveolar macrophage cell line(3D4/21)Mouse Macrophage Cell Line(RAW264.7)
Urinary System
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Mouse prostate cancer cell line(RM-1)Rat adrenal pheochromocytoma cell line(PC-12)Distal nephron cell line(Distal nephron cell line(JU4s))Dog Kidney Cell Line(MDCK)Human prostate cancer cell line(22RV1)Human Embryonic Kidney Cell Line(293T)Human Embryonic Kidney Cell Line(HEK293)Human Bladder Transitional Cell Carcinoma Cell Line(T24)Human bladder carcinoma cell line(5637)Human bladder carcinoma cell line(TCCSUP)
Digestive System
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Human colon adenocarcinoma cell line(LS174T)Porcine intestinal epithelial cell line(IPEC-J2)Human hepatoma cell line(HepaRG)Mouse Hepatocarcinoma Cell Line(Hepa 1-6)Mouse Hepatocarcinoma Cell Line(H22)Human Hepatoma Cell Line(SMMC-7721)Human renal carcinoma cell line(ACHN)African green monkey kidney cell(Vero)Human renal cell carcinoma cell line(786-0)Human Esophageal Squamous Carcinoma Cell Line(KYSE-30)Human Esophageal Squamous Carcinoma Cell Line(KYSE-150)Human gastric cancer cell line(AGS)Human Gastric Cancer Cell Line(SGC-7901)Human Gastric Cancer Cell Line(HGC-27)Human hepatobiliary cancer cell line(RBE)Human hepatocellular carcinoma cell line(HuH-7)Human Hepatoma Cell Line(Hep3B)Human liver cancer cell line(Hep G2)Human Normal Hepatocytes Cell line(L-02)Human colon carcinoma cell line(T84)Human colorectal adenocarcinoma cell line(Caco-2)Human colorectal adenocarcinoma cell line(NCI-H716)Human colon adenocarcinoma cell line(DLD-1)Murine colorectal carcinoma cell line(CT26.WT)Murine Colorectal Carcinoma Cell Line(MC38)Human caucasian colon adenocarcinoma cell line(COLO 205)Human colon carcinoma cell line(RKO)Human Colon Cancer Cell Line(HT-29)Human Colon Cancer Cell Line(SW620)Human Colon Cancer Cell Line(SW480)Human Colon Cancer Cell Line(HCT 116)
Skeleton, Articulus, Soft Tissue, Derma System
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Mouse osteoid cell line(MLO-Y4)Ameloblastoma(hTERT-AM)Mouse squamous carcinoma cell line(SCC7)Mouse myeloma cell line(Sp2/0-Ag14)Human skin squamous carcinoma cell line(A431)Murine melanoma cell line(B16-F10)Human Melanoma Cell Line(M14)Human malignant melanoma cell line(A-375)Human fibrosarcoma cell line(HT-1080)Human bone osteosarcoma epithelial cell line(U-2 OS)Human Osteosarcoma Cell Line(MG-63)
Ocular, Otolaryngologic and Oral System
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Human retinal pigment epithelial cell line(ARPE-19)Human choroidal melanoma cell line(OCM-1)Human oral squamous carcinoma cell line(HSC3)Human Nasopharyngeal Carcinoma Cell Line(C666-1)Human Nasopharyngeal Carcinoma Cell Line(CNE2Z)Human nasopharyngeal carcinoma cell line(NPC-43)Rat Muller Cell Line(rmc-1)
Reproductive System
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Human ovarian cancer cell line(OVCAR-3)Human Villous Trophoblast(HTR-8/SVneo)Human Ovarian Adenocarcinoma Cell Line(CAOV3)Mouse Testicular Stromal Cell Line(TM3)Mouse Pituitary Cell Line(Lbetat2)Human Ovarian Cancer Cell Line(SK-OV-3)Chinese Hamster Ovary Cell Line(CHO-K1)Mouse Embryonic Fibroblasts(NIH/3T3)Human Cervical Carcinoma Cell Line(HeLa 229)Human Cervical Carcinoma Cell Line(HeLa)
Stem Cell
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Stem Cell(H9)Human Embryonic Stem Cell(H1)Induced Pluripotent Stem Cell(ipsc)

PM cells construction Method

4 solutions to meet different mutation needs!
RNP
Prime Editor
Base Editing
Antibiotics-based Konck in

Incubate sgRNA and Cas9 protein in vitro to form an RNP (ribonucleoprotein) complex, which is then co-transfected into cells along with a single-stranded oligonucleotide. After the RNP induces a double-strand break at the target site, the oligonucleotide can introduce the desired mutation into the genomic target through homologous recombination.

Features:

  • Broad applicability
  • High editing efficiency
  • Short turnaround time

Applicable Types:

  • Suitable when a gRNA sequence can be identified within approximately 10 bp of the mutation site.
  • Applicable to target cells that can be transfected using electroporation or liposome-based transfection.

Gene editing is achieved by fusing an engineered reverse transcriptase with a Cas9 nickase and using a special prime editing guide RNA (pegRNA).

Features:

  • Capable of correcting various complex mutations.
  • However, it has low editing efficiency and requires complex vector design.

Applicable Types:

  • Suitable for correcting complex mutations.
  • Ideal for cases where an appropriate gRNA sequence cannot be found near the mutation site.
  • A base editing system is developed by integrating a base deaminase with the CRISPR/Cas system, enabling precise introduction of C/G-to-T/A and A/T-to-G/C point mutations without creating double-strand breaks, thereby achieving highly efficient and accurate gene editing.
  • Features: High efficiency, but applicable cases are limited.
  • Applicable Types: Suitable for specific single-base conversions (C⇌T; A⇌G) when the target site is within the active window.
  • The donor vector is constructed using a plasmid, with a resistance gene expression cassette inserted into an intron to enhance the efficiency of point mutation recombination through antibiotic screening.
  • Features: Suitable for scenarios where a suitable gRNA sequence cannot be identified near the mutation site; however, it is relatively expensive and has a longer experimental cycle.
  • Applicable Types: Ideal for cases where a suitable gRNA sequence cannot be identified near the mutation site.
RNP

Incubate sgRNA and Cas9 protein in vitro to form an RNP (ribonucleoprotein) complex, which is then co-transfected into cells along with a single-stranded oligonucleotide. After the RNP induces a double-strand break at the target site, the oligonucleotide can introduce the desired mutation into the genomic target through homologous recombination.

Features:

  • Broad applicability
  • High editing efficiency
  • Short turnaround time

Applicable Types:

  • Suitable when a gRNA sequence can be identified within approximately 10 bp of the mutation site.
  • Applicable to target cells that can be transfected using electroporation or liposome-based transfection.
Prime Editor

Gene editing is achieved by fusing an engineered reverse transcriptase with a Cas9 nickase and using a special prime editing guide RNA (pegRNA).

Features:

  • Capable of correcting various complex mutations.
  • However, it has low editing efficiency and requires complex vector design.

Applicable Types:

  • Suitable for correcting complex mutations.
  • Ideal for cases where an appropriate gRNA sequence cannot be found near the mutation site.
Base Editing
  • A base editing system is developed by integrating a base deaminase with the CRISPR/Cas system, enabling precise introduction of C/G-to-T/A and A/T-to-G/C point mutations without creating double-strand breaks, thereby achieving highly efficient and accurate gene editing.
  • Features: High efficiency, but applicable cases are limited.
  • Applicable Types: Suitable for specific single-base conversions (C⇌T; A⇌G) when the target site is within the active window.
Plasmid Resistance Method
  • The donor vector is constructed using a plasmid, with a resistance gene expression cassette inserted into an intron to enhance the efficiency of point mutation recombination through antibiotic screening.
  • Features: Suitable for scenarios where a suitable gRNA sequence cannot be identified near the mutation site; however, it is relatively expensive and has a longer experimental cycle.
  • Applicable Types: Ideal for cases where a suitable gRNA sequence cannot be identified near the mutation site.
SNP research needs point mutation cells -Summary of 4 construction methods>>

Knockin Strategies

Protein fusion
The guide RNA and Cas9 complex induce a double-strand break (DSB) at the target site of the DNA. The donor vector carrying the knock-in sequence serves as the template for homologous recombination repair (HDR), facilitating the recombination of the knock-in sequence into the target site.
Protein fusion Knockin cell
Replacement of specific locus:
knockin cell specific locus

Work Flow and Validation

Knockin Cell Line Work Flow and Validatio Knockin Cell Line Work Flow and Validatio

Case Study

A549 Cell MYC Gene - Lung Cancer
IF=20.8
Nature Metabolism
A549 Cell MYC Gene - Lung Cancer
IF=20.8 Nature Metabolism
Acetate reprogrammes tumour metabolism and promotes PD-L1 expression and immune evasion by upregulating c-Myc
Published by:Zhejiang University and the Chinese Academy of Medical Sciences
Abstract:
This study demonstrated that acetate is the most abundant short-chain fatty acid in human non-small cell lung cancer tissue, with increased acetate uptake as the tumors enriched. The study used Ubigene-constructed MYC (p.K148R) point mutant A549 cells and MYC (p.K148Q) point mutant A549 cells, simulating acetylated and non-acetylated c-Myc states, and investigated the impact of these changes on tumor cell behavior. View details>>
Mechanism pattern diagram
Figure 1 Mechanism pattern diagram
FOXP1 gene in HEK293 cells - Tumor occurrence
IF=9.4
The EMBO Journal
FOXP1 gene in HEK293 cells - tumor occurrence
IF=9.4 The EMBO Journal
Abstract:
This study reveals a new mechanism by which the forkhead box (FOX) transcription factor FOXP1 promotes ATR-CHK1 activation under replication stress. This function does not rely on the transcriptional regulatory activity of FOXP1 but is instead co-regulated by its phosphorylation and glycosylation modifications. The study used the source cell lines from Genewell to construct the FOXP1 gene S396A and S396D point mutation HEK293 cell lines.
Mechanism pattern diagram
Figure 1 Mechanism pattern diagram

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