Home -> Cell Biology -> Stem Cell related Products -> Pluripotency Induction: Integrase based

Pluripotency Induction: Integrase based

The phiC31 Integrase System - Nonviral technology for transgenesis

• All-in-one iPSC Vector
• Works in All Model Organisms
• Validated Mouse Colonies in One Month
• Single Copy Integration
• No Gene Size Limit
• Removable with FLP Recombinase
• Highly efficient

The phiC31 integrase is a site-specific recombinase encoded within the genome of the bacteriophage phiC31. The phiC31 integrase mediates recombination between two ~34 base pair sequences termed attachment sites (att), one found in the phage and the other in the bacterial host. This serine integrase has been show to function efficiently in many different cell types including mammalian cells. In the presence of phiC31 integrase, an attB containing donor plasmid can be unidirectional integrated into a target genome through recombination at sites with sequence similarity to the native attP site. These sites are termed pseudo-attP sites. phiC31 integrase can integrate a plasmid of any size, as a single copy, and requires no cofactors. The integrated transgenes are stably expressed and heritable.

Image


phiC31 Integrase iPSC Vectors

The classic Oct3/4, Sox2, Klf4 and cMyc factors have been adapted to the integrase system. The pCOBLW phiC31 reprogramming vector features the iPSC factors driven by a potent CAGs promoter with the factors separated by P2A elements. HS4 insulators flank the expression cassette and a functional attB site directs the integration of the entire plasmid into pseudo attP sites in any genome. The vector is engineered with FRT sites for later excision using standard FLP recombinase methods. After co-transfection of the reprogramming vector with the phiC31 expression plasmid (catalog# FC200PA-1) and integration, you can monitor for successful transfection with GFP fluorescence and select for positive colonies using the built-in Neo marker. The reprogramming iPSC factor donor vector and the phiC31 expression plasmid maps are shown below.

Image

Integration Analysis

A major advantage of using the phiC31 integrase system, is that copy number control is in your hands. The majority of clones that undergo integration events have single insertions. To verify the copy numbers in your targeted cells, you can perform Southern blot analysis on their genomic DNA. Shown below is an example of mouse genomic integration analyses using seperate restriction digests with Eco01091 and EcoRV with 10ug gDNA. The reason for two separate enzyme digests is to assess the copy number status. The probe used in these Southern blot analyses was for GFP present in the pCOBLW iPSC integrase vector. The data below show the copy number status for 16 different iPSC lines and demonstrate that 14 out of 16 lines show single insertions (88%).

Image


References

Karow M, Chavez CL, Farruggio AP, Geisinger JM, Keravala A, Jung WE, Lan F, Wu JC, Chen-Tsai Y, Calos MP. Site-specific recombinase strategy to create induced pluripotent stem cells efficiently with plasmid DNA. Stem Cells, 2011 Nov, 29 (11): 1696-704.

Chalberg TW, Portlock JL, Olivares EC, Thyagarajan B, Kirby PJ, Hillman RT, Hoelters J, Calos MP. Integration specificity of phage phiC31 integrase in the human genome. J Mol Biol. 2006 Mar 17;357(1):28-48.

Takahashi, K. and Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663-676.

Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka S. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nature Biotechnology. 26: 101-106.

Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells. Science 318: 1917-1920.

Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature. 448:313-7.

Related Links

phiC31 Integrase Vector System

 

 

Description Cat# Size Price    
Non-Viral PhiC31 Integrase Mouse iPSC Reprogramming System (includes FC305A-1 and FC200PA-1) FC300A-1-SBI 2 x 10 ug 1742 € DETAILS   Add to Cart 
pCOBLW [CAG-Oct4-Sox2-Klf4-Myc-GFP-SV40-Neo] Mouse Reprogramming PhiC31 Donor Plasmid FC305A-1-SBI 10 ug 1676 € DETAILS   Add to Cart 
PhiC31 Integrase Expression Plasmid FC200PA-1-SBI 10 ug 610 € DETAILS   Add to Cart 

BioCat Special

% Special Offers

Benefit from our current promotions

SARS-CoV-2 / COVID-19

Tools for SARS-CoV-2 Resarch

Electrophoresis

30% OFF BioCat Universal Agarose

Email Newsletter

Subscribe to the BioCat Email Newsletter.

Logos

Logos Biosystems Cell Counters

Cell-based Assays

Cell-based assays

Choose from a wide variety of cell-based assays designed for greater accuracy and increased throughput.

50% OFF Fluorescent Proteins

Fluorescent Protein Vectors

Get 50% discount on the second and subsequent vectors when ordering two or more vectors with the same fluorescent protein.

Lentiviral Barcode Library

Lentiviral-based barcode library

Track clonal populations of cells over time or through different treatments using the lentiviral-based barcode library.

Home
Imprint / Impressum | Privacy Policy / Datenschutzerklärung
Top of Page Up!