Creative Bioarray can successfully construct stably transfected cell lines with GOI site-specific integration. Use specific integrated enzymes in specific locations to achieve related needs and provide our customers with one-stop solutions. We have established a mature agreement to provide stable cell line products that can be edited at fixed points according to customer needs.
Mammalian cell expression systems are widely used to produce biotherapeutic proteins, but their development is often hindered by the unstable and variable transgene expression associated with random integration. This is especially evident in the development of CHO cells. The heterogeneity in the cell bank caused by random integration requires tedious screening of multiple clones to identify those clones with suitable production characteristics. In contrast, the SSI system, where a transgene encoding a recombinant protein of interest is integrated into a predetermined genomic locus, provides a way to generate more consistent clones, thereby reducing cell line development time. Therefore, SSI has become a promising strategy through which the development of stable transgenic cell lines can repeatedly target its preferred genomic locus with high activity and stable expression.
The cell line characteristics directly related to the transgene integration site, such as expression level and stability, are called positional effects. The transcriptional activity of a specific site is controlled by its chromatin state and corresponding accessibility. Chromosomal loci that have the inherent ability to confer special stability and enhance transcriptional activity are called hot spots. It has been reported that the hot spot for increased recombinant protein expression (HIRPE) was found to trigger the formation of inverted repeats and contain several matrix attachment regions (MAR)-like sequence elements. When included in the transfected rDNA sequence, the MAR linking chromatin to the nuclear matrix has previously been shown to increase transgene expression levels. Site-specific recombinase (SSR)-mediated SSI provides a more aggressive method for genomic hot spot identification.
Fig 1. Nuclease-mediated approaches for SSI in CHO cells. (Hamaker N K, et al. 2018)
The mammalian cell expression system is the most commonly used platform for the production of biotherapeutic proteins. However, the development of mammalian recombinant cell lines is often affected by unstable and variable transgene expression associated with random integration. Our site-specific integration stable cell line development employs an effective strategy for the site-specific integration of genes of interest (GOI). This strategy is based on the SSR-based system to quickly and accurately insert the gene expression cassette at a specific site in the host cell, thereby achieving homogeneous transgene expression. Our site-specific integration systems all support unlimited insertion sizes, site-specific integration and provide various promoter and reporter combinations.
| Optional Content | Description |
|---|---|
| Gene-directed Mutation | Make precise mutations to one or several bases of the target gene; |
| Gene-directed Integration | The target fragment can be integrated into a specific location on the genome; |
Fig 2. Our site-specific integrated stable cell construction service process.
| SSI Method | Advantages |
|---|---|
| Recombinase-mediated |
✓ Useful for empirical identification of hot spots； ✓ Amenable to negative selection and promoter trap； ✓ Generates retargetable master cell line platform； |
| Nuclease-mediated |
✓ Targets predetermined chromosomal coordinates with known expression characteristics； |
| Hybrid (nuclease- and recombinase-mediated) |
✓ Targets predetermined chromosomal coordinates； ✓ Amenable to negative selection and promoter trap； ✓ Generates retargetable master cell line platform； |
Creative Bioarray provides technical services from vector construction to the formation of stable transfected GOI silent cell lines, helping our customers save time and effort. You will benefit from our technical expertise and state-of-the-art facilities and work with our scientific team to find the best solution to meet your needs.
If you are interested in our services or have any specific needs, please feel free to contact us. We look forward to working with you in the near future.
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