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Antibody-Producing Cell Lines Development


Currently, stable cell lines have been widely used in various research applications, including recombinant protein...

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Antibody-Producing Cell Lines Development

Currently, stable cell lines have been widely used in various research applications, including recombinant protein and antibody production, screening of experimental drugs, functional study of gene, assay development and so on.

Therapeutic antibodies are mainly produced in mammalian host cell lines including NS0 murine myeloma cells and Chinese hamster ovary (CHO) cells [1]. CHO cells are the predominant host used to produce therapeutic proteins. About 70% of all recombinant proteins produced today are made in CHO cells [2]. It is important to develop a stable antibody-producing cell line to meet the high dosage requirement of therapeutic antibodies for the growing cancer patient population.

Our scientists have years of experience at performing gene editing with CRISPR/Cas9, and have generated series of gene knockout CHO-K1 cell lines developed by CRISPR/Cas9 system. Our gene editing CHO-K1 cells can save your precious time and effort with great performance in antibody production.

We have a list of ready to use CHO-K1 cells targeting genes crucial in antibody production process.

1. Fut8 Knockout- Increasing Antibody-Dependent Cellular Cytotoxicity (ADCC)

Fut8 encodes an enzyme belonging to the family of fucosyltransferases. The product of this gene catalyzes the transfer of fucose from GDP-fucose to N-linked type complex glycopeptides. FUT8(-/-) CHO-K1 cells which increases antibody-dependent cellular cytotoxicity (ADCC) has been widely used as hosts for antibody production.

2. GS Knockout- Rapidly Selection of CHO-K1 Cell Lines for High Level Antibody Production

GS encodes an enzyme belongs to the glutamine synthetase family. Glutamine is an abundant amino acid, and is important to the biosynthesis of several amino acids, pyrimidines, and purines. Mutations in this gene are associated with congenital glutamine deficiency. Glutamine Synthetase (GS) Expression System can be used for rapidly selection of CHO-K1 cell lines for high level antibody production.

3. Bax/Bak Knockout- Resistant to Apoptosis

Programmed cell death, which is required for the development and homeostasis of metazoans, includes mechanisms such as apoptosis, autophagic cell death, and necrotic (or type III) death. Members of the Bcl2 family regulate apoptosis, among which Bax and Bak act as a mitochondrial gateway. Bax/Bak double-knockout mice are resistant to apoptosis [3].

Advantages

  1. Rapidly selection of CHO cell lines using the Glutamine Synthetase (GS) Expression System for high level antibody production
  2. Increasing antibody-dependent cellular cytotoxicity (ADCC)
  3. Excellent performance with high antibody production, high antibody quality, or a short timeline

Antibody-Producing Cell Line Product List

Cat.Product NameTarget GeneHost Cell
CSC-RT0097GS/Fut8 Knockout Cell Line-CHO-K1GS/Fut8CHO-K1
CSC-RT0103GS/Bax/Bak1 Knockout Cell Line-CHO-K1GS/Bax/Bak1CHO-K1
CSC-RT0119GS/BAX Knockout Cell Line-CHO-K1GS/BAXCHO-K1
CSC-RT0125GS Knockout Cell Line-CHO-K1GSCHO-K1

References:

  1. Xu N, Ou J, Gilani A K A, et al. High-level expression of recombinant IgG1 by CHO K1 platform[J]. Frontiers of Chemical Science and Engineering, 2015, 9(3): 376-380.
  2. Li F, Vijayasankaran N, Shen A, et al. Cell culture processes for monoclonal antibody production[C]//MAbs. Taylor & Francis, 2010, 2(5): 466-479.
  3. Arakawa S, Tsujioka M, Yoshida T, et al. Role of Atg5-dependent cell death in the embryonic development of Bax/Bak double-knockout mice[J]. Cell Death & Differentiation, 2017.

Case Study I

CSC-RT0125    GS Knockout Cell Line-CHO-K1

  1. Design gRNA targeting GS gene and construct knockout vector.
  2. CHO-K1 cells were transfected with the construct and the cell pool was analyzed by Sanger sequencing.
  3. Digest PCR products with our knockout detection (KOD) enzyme to select positive clones.
  4. TA cloning and sequencing. A single clone containing a 32 bp deletion in allele 1 and allele 2 was carried forward (Fig. 1).
  5. To assess loss of function, GS knockouts were grown in the presence or absence of increasing concentrations of glutamine (Fig. 2).

Fig. 1 Sequencing verification of TA cloning

Fig. 2 L- glutamine Dependence of CHO-K1 (GS-/-)

GS knockouts were unable to grow in the absence of glutamine. However, growth improved in the presence of increasing concentrations of glutamine, thereby indicating a functional loss in the GS knockout cell line.


Case Study II

CSC-RT0097    GS/Fut8 Knockout Cell Line-CHO-K1

  1. Design gRNA targeting Fut8 gene and construct knockout vector.
  2. CHO-K1-GS-/- cells (CSC-RT0125) were transfected with the construct and the cell pool was analyzed by Sanger sequencing. (Fig. 1)
  3. Digest PCR products with our KOD enzyme to select positive clones. (Fig. 2)
  4. Individual clones were Sanger sequenced to select for homozygous knock-out indels of Fut8. (Fig. 3)

Fig. 1 The sequencing chromatogram of PCR product for positive clone

Fig. 2 Digestion of PCR products with KOD enzyme

KOD enzyme can recognize the nicks resulted from the knocking out process and will therefore cut such PCR products into two bands.

Fig. 3 Sequencing verification of TA cloning
(11 bp deletion in allele 1 and 1 bp insertion in allele 2)

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