KO/KI Cells

Process of generating knock-out and knock-in cells

PNA Bio offers economic cell line generation (knock-outs and knock-ins) in a timely manner. The process of engineered cell line generation is shown below.

  • Design and synthesis of 4~8 gRNA vectors
  • Select the most efficient engineered nuclease by mismatch sensitive nuclease assay
  • Synthesis of reporter vector for the enrichment of cells with gene editing event
  • Optimization of transfection protocol
  • Design of donor vector for knock-in project
  • Selection of the pool by MACS, FACS, or Hygromycin resistance (option: efficiency validation in a selected pool)
  • Single cell cloning
  • Screening of the clones using T7E1 assay and/or F-PCR
  • Confirmation of homozygous knock out by TA cloning and sequencing

Advantage of our service

  • Years of experience using ZFN, TALEN, and CRISPR/Cas9 system
  • In house developed software to design gRNA sequences with maximum activity and minimum off-site effect
  • Robust assay system to screen and select the most optimal engineered nucleases
  • Reporter system to enrich cells with gene editing (3~20 fold improvement over conventional method)

Some of the cell lines that we have generated knock-outs or knock-ins

  • Commonly used cells: HEK293, NIH3T3, CHO cells
  • Human cancer cells: HeLa, HCT116, SKBR3, Hep3B, HepG2, MCF7, K-562, H-358 cells
  • Mouse cells: Pan02, CT26WT cells
  • Neuronal cells: Sy5y, PC12 cells

We have successfully generated knock-out clones up to 5 copies in one round.

Examples of knock-out clones generated

CRISPR/Cas9 knock out KO cell line generation
Example of KO clone generation: sequencing analysis of two KO clones and T7E1 analysis for five off-target sites that contain three mismatches.

We offer the most affordable engineered cell line service in a timely manner.

Knock-out cell line service

  • Provide at least two independent KO clones, confirmed for mycoplasma free
  • Time line: 4~7 months

Knock-in cell line service

  • Provide at least two independent clones, confirmed for mycoplasma free
  • Includes donor vector design and synthesis
  • Time line: 5~7 months
  • Confirmation of KO by FACS (example)

    Knock-out KO cell line generation

For quotation, please send us an email with a gene name, cell line, and the goal of your project to order@pnabio.com or click here.

We usually respond within 24 hours after the inquiry was received. Also feel free to call at 805) 277-0227 for consultation over the phone.

© 2022 PNA BIO INC All Rights Reserved | "We Guarantee Excellent Quality for All Our Products"