Creation of Custom Cell Lines
Construction of stable gene expression cell lines
Artificial chromosome vectors have various features not found in the conventional gene expression system. We provide services for the construction of stable gene expression cell lines using these features. Stable gene expression cell lines can be used for the screening of drug candidate compounds and in vitro pharmacology tests in the drug discovery process.
| Problems in existing expression systems | Advantages of using artificial chromosome vector | |
|---|---|---|
| Gene loading | ・Limited mounting size ・Restriction enzyme restriction site and size restrictions due to gene incorporation by restriction enzyme |
・Since there is no limit on the size to be mounted, genomic DNA can be loaded (splicing variant can be evaluated) ・Multiple genes can be loaded (protein – protein interaction analysis etc can be done) |
| promoter | ・In the case of forced expression by an exogenous promoter, the expression level can not be controlled (possibility different from physiological condition) | ・Expression by the original promoter is possible, so expression under physiological conditions is possible (expression amount reflecting living body or tissue specific expression can be performed) |
| Construction of gene stable expression cell line | ・For constitutive expression, it must be inserted into the host chromosome ・The site to be inserted, the number of copies inserted are various ・Overexpression or silencing occurs because it is affected by the site of insertion into the host chromosome |
・It exists independently from the host chromosome ・The same artificial chromosome vector can be transferred into various cell lines ・Expression is not affected by the host chromosome. |
| Vector stability | ・Expression loss may occur | ・Maintained with constant copy number for long periods |
| Mounted gene | DNA type | Recipient cell | Overview | Cited document |
|---|---|---|---|---|
| CMV-human EPO | cDNA | HFL-1 | Construction of erythropoietin expression system | Kakeda et al., 2005(17) |
| CAG-human FVIII | cDNA | CHO hprt-, hiMSC | Expand 1-16 copies of FVIII gene | Kurosaki et al. 2011(11) |
| HSP70-insulin | cDNA | HT1080 | Expression of insulin using HSP70 promoter | Suda et al 2006(14) |
| Human dystrophin | genome | hiMSC,mouse,mdx-iPS, DMD-iPS | Introduction of HAC vector containing dystrophin gene into IPS cells | Hoshiya et al. 2009(20) Kazuki et al. 2010(18) |
| Mouse CD40L | genome | Jurkat,U937 | Expression of CD40 ligand gene | Yamada et al. 2008(13) |
| Human P53 | genome | mGS p53-/- | Introduction of hP53 gene into mouse p53-deficient cells | Kazuki et al. 2008(23) |
| TR-DNA-PKcs | cDNA | V3 | Expression control of DNA-PKcs related to gene repair by Tet system | Otsuki et al 2005(16) |
| Ubc-hTERT-IRES-GFP | cDNA | HFL-1 | Extension of the life span of normal fibroblasts by expression of telomerase reverse transcriptase gene | Shitara et al. 2008(26) |
| OPN-EGFP | cDNA | hiMSC | Construction of expression system specific to cell type by OPN promoter | Ren et al. 2005(25) |
| OC-GFP | cDNA | CHO | Screening system for foods etc. containing osteogenic factor by expression of GFP from OC promoter | Takahashi et al 2010(27) |
Excerpt from Kazuki Y et al., Gene Ther 2011 Table 2
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Please contact us for quotation about cell construction with artificial chromosome vector.