cell assays

Cell Based Assays in Cell Cycle II

The C-terminal region of human DNA helicase B (HDHB) is a 99 amino acids polypeptide that contains both a putative nuclear localization signal (NLS) and a nuclear export signal (NES). It undergoes cell cycle-dependent translocation. GFP-HDHBCT is a fusion of GFP and the C-terminal region of HDHB. It remains inside the nucleus during G1 phase, then translocates into and remains in the cytoplasm during S and G2 phases. After nuclear envelope breaks down in M phase, this biosensor is localized throughout the cell. After nuclear envelope reformation at the end of M phase, the G1 phase biosensor is imported into the nucleus.

Hahn AT, Jones JT, Meyer T. Quantitative analysis of cell cycle phase durations and PC12 differentiation using fluorescent biosensors. Cell Cycle. 2009 Apr 1;8(7):1044-52. Epub 2009 Apr 2.

This biosensor is a fusion protein comprising three components, a reversible plasma membrane–targeting domain fused to the N terminus of enhanced yellow fluorescent protein (EYFP), which is in turn fused to the N terminus of a nuclear localization signal (NLS). The nuclear localization signal would anchor the reporter’s localization to the nucleus during the G1, S and G2 phases (interphase) of the cell cycle. Upon the nuclear envelope breakdown at the onset of prometaphase, the plasma membrane–binding domain would cause reversible translocation of the biosensor to the plasma membrane where the fluorescence could be monitored by microscopy.

Jones JT, Myers JW, Ferrell JE, Meyer T. Probing the precision of the mitotic clock with a live-cell fluorescent biosensor. Nat Biotechnol. 2004 Mar;22(3):306-12.

E2F-mClaverGR TRE Reporter
The E2F family of transcription factors is a key regulator of cell-cycle checkpoints in mammalian cells. The E2F protein is a major target of the retinoblastoma gene product (Rb) and the activity of E2F/pRb is intimately connected with the G1-S transition of the cell cycle. The E2F protein forms a heterodimer complex with DP1, which binds to E2F response elements and initiate transcription.
The E2F-responsive mClaverGR construct encodes a photoconvertible green-to-red fluorescent protein reporter gene under the control of a minimal (m)CMV promoter and tandem repeats of the E2F transcriptional response element (TRE). We have experimentally optimized the number of response elements as well as the intervening sequence between response elements to maximize the signal to noise ratio.

Ki67, encoded by MKI67 gene, is expressed in all phases of the active cell cycle (G1, S, G2 and M phase), while is absent in the resting phase (G0). Therefore, it is routinely used as a marker of cell cycling and proliferation. Recently, Zambon AC[1] cloned and characterized the 1.5 kb proximal promoter (Ki67p) of the human Ki67 gene. A reporter, Ki67p-GFP, was further constructed to express GFP under Ki67p. Their data verified that GFP driven by Ki67p is co-expressed in cells with endogenous Ki67 and is correlated with cells transitioning through G1/S/G2/M phases of the cell cycle. Mitomycin C induced G1/S/G2/M blocking or cell-density induced cell cycle arrest both attenuate Ki67p activity.

Zambon AC. Use of the Ki67 promoter to label cell cycle entry in living cells.Cytometry A. 2010 Jun;77(6):564-70.

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Wednesday, November 3rd, 2010 Fluorescent proteins 1 Comment

Allele Custom Services for Drug Screening Companies

Many target discovery and validation programs can benefit from RNA interference, fluorescent proteins, stem cells, and viral delivery systems. However, applications of these technologies require special reagents and laboratory know-how. Even when available, many generic reagent kits are not tailored for your particular needs in screening or validation.

At Allele, we accelerate your discovery efforts with custom RNAi screening, fluorescence based assays, and cell model development services.

1) Our RNAi platform, based on our patented shRNA/miRNA technologies, use DNA linear template, plasmid, lentivirus, retrovirus, or baculovirus vectors that prompt cells to endogenously express RNAi. As a result, our screens offer advantages over synthetic siRNAs:
• Higher levels of consistency
• Greater delivery and gene silencing efficiencies
• Accessibility to difficult-to-transfect cells, including primary cells
• Potential for inducible RNAi expression
• More persistent silencing with shRNA under Allele’s own IP–you may not need to license siRNA patents!

2) Fluorescent proteins (FPs), which can span the entire visual spectrum, have become some of the most widely used genetically encoded tags. Genes encoding FPs alone or as fusions to a protein of interest may be introduced to cells by a number of different methods, including simple plasmid transfection or viral transduction. Allele Biotech is one of a few companies that develop and improve FPs through fundamental research. We have so far achieved:
• The brightest cyan and green FPs, true monomers for minimum artifact or cytotoxicity
• The brightest yellow and red FPs from lancelet, only FPs from vertebrate
• mTFP1 as the best FRET donor by 3 independent reports
• Photoconvertible FPs for super imaging or kinetic labeling
• Delivery on plasmid, retrovirus, lentivirus, baculovirus vectors

3) As a major advancement in the stem cell field, it has recently been shown that mouse and human differentiated cells may be reprogrammed into stem-like, pluripotent cells by the introduction of defined transcription factors. These induced stem cells (iPSCs) provide unprecedented resources of cells of different differentiation stages for functional testing and drug screening. Allele Biotech develops and provides state-of-the-art reagents in convenient forms for iPSC production
• iPS factors carried on lentivirus, retrovirus, baculovirus for different cell types
• Availability in combination with fluorescent proteins under own IP, and drug resistant genes
• 4-in-1 or 2-in-1 effective use of iPS factors on one viral vector
• Feeder cells of human origin expressing factors essential for stem cell culturing

4) Introduction of protein factors, miRNA, promoter-reporter, and virtually any other genetic element of interest via the most efficient viral packaging systems.
• Introducing protein-FP fusion, promoter-FP reporter, photoactivatable factors for cell-based assays
• Introducing critical factors for cell immortalization
• Episomal or integrated expression using baculoviral vectors
• High throughput, systematic expression of whole class of molecules in any type of cell
• High titer viral packaging at low cost for delivery to animal tissues

In addition, the Allele team can provide custom-designed assays that can be used for assaying enzyme activities in almost any pathway, such as the EGF pathway, TNF response/apoptosis pathway, nuclear receptors, etc. We utilize technically advanced methods to provide our partners with advantages over alternative methods or other services.

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Wednesday, June 30th, 2010 Open Forum, RNAi patent landscape No Comments