pcDNA6.2/nGeneBLAzer-GW/D-TOPO载体含有以下元件: Human cytomegalovirus immediate-early (CMV) promoter/enhancer for high-level expression in a wide range of mammalian cells
β-lactamase bla(M) reporter gene for C-terminal (pcDNA6.2/cGeneBLAzerGW/D-TOPO) or N-terminal (pcDNA6.2/nGeneBLAzer-GW/D-TOPO)fusion to the gene of interest
attB1 and attB2 sites for site-specific recombination of the expression clone with a Gateway donor vector to generate an entry clone
Directional TOPO Cloning site for rapid and efficient directional cloning of blunt-end PCR products (see page 3 for more information)
The V5 epitope tag for detection using Anti-V5 antibodies (pcDNA6.2/nGeneBLAzer-GW/D-TOPO only)
The Herpes Simplex Virus thymidine kinase polyadenylation signal for proper termination and processing of the recombinant transcript
f1 intergenic region for production of single-strand DNA in F plasmidcontaining E. coli
SV40 early promoter and origin for expression of the Blasticidin resistance gene and stable propagation of the plasmid in mammalian hosts expressing the SV40 large T antigen
Blasticidin resistance gene for selection of stable cell lines
The pUC origin for high copy replication and maintenance of the plasmid in E. coli
The ampicillin resistance gene for selection in E. coli GeneBLAzer技术的优势 Suitable for use as a sensitive reporter of gene expression in living mammalian cells using fluorescence microscopy.
Provides ratiometric readout to minimize differences due to variability in cell number, substrate concentration, fluorescence intensity, and emission sensitivity.
Compatible with a wide variety of in vivo and in vitro applications including microplate-based transcriptional assays and flow cytometry.
Provides a flexible and simple assay development platform for gene expression in mammalian cells.
Using a non-toxic substrate allows continued cell culturing after quantitative analysis. GeneBLAzer系统的组成 The GeneBLAzer System facilitates fluorescence detection of β-lactamase reporter activity in mammalian cells, and consists of two major components:
The β-lactamase reporter gene, bla(M), a truncated form of the E. coli bla gene.
When fused to a gene of interest, the bla(M) gene can be used as a reporter of gene expression in mammalian cells. For more information about the bla(M) gene, see below.
A fluorescence resonance energy transfer (FRET)-enabled substrate, CCF2 to facilitate fluorescence detection of β-lactamase activity. In the absence or presence of β-lactamase reporter activity, cells loaded with the CCF2 substrate fluoresce green or blue, respectively. Comparing the ratio of blue to green fluorescence in a population of live cells or in a cell extract of your sample to a negative control provides a means to quantitate gene expression. For more information about the CCF2 substrate and how FRET works, refer to the GeneBLAzer Detection Kits manual. β内酰胺酶基因(bla) β-lactamase is the product encoded by the ampicillin resistance gene (bla) and is the bacterial enzyme that hydrolyzes penicillins and cephalosporins. The bla gene is present in many cloning vectors and allows ampicillin selection in E. coli. β-lactamase enzyme activity is not found in mammalian cells.bla(M) Gene The GeneBLAzer Technology uses a modiied bla gene as a reporter in mammalian cells. This bla gene is derived from the E. coli TEM-1 gene present in many cloning vectors (Zlokarnik et al., 1998), and has been modified in the following ways:
72 nucleotides encoding the first 24 amino acids of β-lactamase were deleted from the N-terminal region of the gene. These 24 amino acids comprise the bacterial periplasmic signal sequence, and deleting this region allows cytoplasmic expression of β-lactamase in mammalian cells.
The amino acid at position 24 was mutated from His to Asp to create an optimal Kozak sequence for optimal translation initiation. This modified reporter gene is named bla(M).
Note: The TEM-1 gene also contains 2 mutations (at nucleotide positions 452 and 753) that distinguish it from the bla gene in pBR322 (Sutcliffe, 1978). 检测重组蛋白 Depending on the kit you are using, you will assay for β-lactamase reporter activity through in vivo or in vitro detection methods. A brief description of each detection method is provided below. For detailed information, refer to the GeneBLAzer Detection Kits manual. If you have generated a pcDNA6.2/nGeneBLAzer-GW/D-TOPO expression construct that contains your gene of interest fused to the V5 epitope tag, you may also detect your recombinant fusion protein by Western blot analysis using the Anti-V5 Antibodies. 体外检测 Using the GeneBLAzer In Vitro Detection Kit allows you to quantitate the amount of intracellular β-lactamase in cells based on the β-lactamase activity in lysates.
To detect β-lactamase activity in mammalian cell lysates, use the CCF2-FA substrate. CCF2-FA is the non-esterified, free acid form of CCF2, and is recommended for in vitro use because it is readily soluble in aqueous solution and may be added directly to pre-made cell lysates. Once added to cell lysates, you may quantitate the CCF2-FA fluorescence signal using a fluorescence plate reader or a fluorometer.
To prepare cell lysates from mammalian cells containing the bla(M) reporter gene, you must use a method that will preserve the activity of the β-lactamase enzyme.
Refer to the GeneBLAzer Detection Kits manual for detailed guidelines and protocols to prepare CCF2-FA solution, prepare cell lysates and samples, and detect CCF2 signal. 体内检测 Using the GeneBLAzer In Vivo Detection Kit allows you to measure β-lactamase reporter activity in live mammalian cells. Once β-lactamase reporter activity has been measured, cells may cultured further for use in additional assays or other downstream applications.
To detect β-lactamase activity in live mammalian cells, use the CCF2-AM substrate. CCF2-AM is the membrane-permeable, esterified form of CCF2, and is recommended for in vivo use because it is non-toxic, lipophilic, and readily enters the cell. Once cells are “loaded” with CCF2-AM, you may quantitate the CCF2 fluorescence signal using a variety of methods.
Refer to the GeneBLAzer Detection Kits manual for detailed guidelines and protocols to prepare CCF2-AM solution, load cells with CCF2-AM substrate, and detect CCF2 signal. 实验材料： Purified plasmid DNA of your entry clone (50–150 ng/ μL in TE, pH 8.0)
pcDNA6.2/nGeneBLAzer-GW/D-TOPO or pcDNA6.2/nGeneBLAzer-GW/D-TOPO vector (150 ng/μL in TE, pH 8.0)
LR Clonase enzyme mix (keep at –80°C until immediately before use)
5X LR Clonase Reaction Buffer (supplied with the LR Clonase enzyme mix)
pENTR-gus positive control, optional (50 ng/μL in TE, pH 8.0; supplied with the LR Clonase enzyme mix)
TE Buffer, pH 8.0 (10 mM Tris-HCl, pH 8.0, 1 mM EDTA)
2 μg/μL Proteinase K solution (supplied with the LR Clonase enzyme mix; thaw and keep on ice until use)
Appropriate competent E. coli host and growth media for expression
S.O.C. Medium
LB agar plates containing the appropriate antibiotic to select for expression clones
