0
Transcription Factor oligodeoxynucleotide decoys (ODN decoys). Use our GeneDetect® transcription factor decoys to inhibit specific transcription factors in cell culture. Complete list of Transcription Factor decoy Products. Introduction
Transcription factor ODN decoy approach
Advantages and disadvantages of the ODN decoy approach for studying cellular gene expression
ODN Decoys available from GeneDetect.com
How are these decoys used?
Controls
References
Introduction
Cells can respond to stimuli (normal or pathological) by changing the levels of expression of specific genes. The cellular proteins that regulate changes in gene expression are called transcription factors. Transcription factors are generally nuclear and can either be constitutively expressed within the cell (present under basal conditions, for example CREB) or themselves inducible (for example AP-1). These transcription factor proteins bind specific sequences found in the promoter regions of genes (target genes) whose expression they then regulate (switch on or off). These binding sequences are generally 6-10 base pairs in length and are occasionally found in multiple copies within the promoter regions of target genes. Although the transcription factor protein-DNA interaction is sequence-specific, the binding site for one given transcription factor may vary by several base pairs within different target genes. Therefore when we describe the specific DNA binding sequence for a transcription factor we refer to the non-variable part of the binding sequence, that is, the transcription factor consensus sequence. For example, the AP-1 transcription factor made up of Fos and Jun proteins binds to the TGACTCA consensus sequence. In comparison the consensus sequence for the Smad transcription factor family which mediate TGF-b, activin and BMP induced changes in gene expression is CAGACA. Fig. 1. Transcription factor ODN decoy approach. The basic theory behind the transcription factor ODN decoy approach involves flooding the cell with competing synthetic, transcription factor-specific consensus sequences. These synthetic decoys "compete" for binding of the transcription factor with consensus sequences in target genes. If delivered into the cell in sufficient concentrations these "decoys" thus have the potential to attenuate the binding of the transcription factor to promoter regions of target genes and thus attenuate the function of the transcription factor to regulate the expression of its target gene(s). Transfected at high concentrations these decoys have been reported in the literature to completely block transcription factor function. Clearly they represent powerful research tools for studying gene regulation both in vitro and also more recently in vivo (for Reviews see Moshita et al., 1998, Mann and Dzau, 2000). Fig. 2. Advantages and disadvantages of the ODN decoy approach for studying cellular gene expression. Advantages. # ODN decoys offer a means of specifically inhibiting transcription factor function in living cells. # Inexpensive compared to other more classical methods of investigating gene expression such as chloramphenicol acetyltransferase and luciferase constructs in promoter-reporter gene transfection experiments. # Allows for investigation of both endogenous and pathological gene regulation # Proven to be highly effective and selective within in vitro experiments. # Easy to use. Disadvantages. # An emerging technology that has not yet been fully characterized # Issues of decoy synthesis. High levels of purity and stability required. # Transfection issues. Which method is best. How to optimize transfection. # Issue of controls. What controls are needed. ODN Decoys available from GeneDetect.com Click here for a full listing. We have designed ODN decoys to over 45 common transcription factors. Our decoys are double-stranded synthetic phosphorothioate deoyxynucleotides which range in length from 20-28 base pairs. The transcription factor consensus sequence occurs within the middle of the decoy sequence and is flanked by carefully selected base-pairs that allow for "optimized" transcription factor binding. These ODN decoys are also available labeled so that you are able to optimize your specific transfection technique by imaging the passage of the decoy into the cell (for example by fluorescence microscopy). Our ODN decoys are purified by HPLC and assessed by gel electrophoresis to ensure that >99% of decoy supplied represents full length, double stranded, functional decoy. As a control, matching mutant decoys are available for each transcription factor. Mutant decoys have the same flanking sequences but contain a disrupted consensus sequence in comparison with the (wild type) ODN decoy. How are these decoys used?The majority of experiments to date have used transcription factor ODN decoys to examine gene regulation in cultured primary cells and cell lines. The most important variables involved in determining whether or not your ODN decoy performs its required function include (a) the combination of cell type/cell density and transfection reagent used (b) the time cells are incubated with ODN decoy and (c) the concentration of ODN decoy used. A. The combination of cell type/cell density and transfection reagent used. While some investigators have achieved success by simply adding naked ODN decoys directly into the cell culture media, the most common method of in vitro ODN decoy transfection is to mix the ODN decoy with a cationic lipid to form a liposome complex before adding the ODN decoy/liposome mixture directly to the media. To aid transfection therefore we recommend mixing of your ODN decoy with an effective liposome-based carrier substance. One transfection reagent we have had good success with in our laboratories is the OligofectAMINE reagent. This is a proprietary formulation available from Invitrogen that is designed to optimize transfection of phosphorothioate ODNs into eukaryotic cells. Stable complexes are formed between the lipid and the ODN permitting efficient delivery of the ODN into mammalian cells. This product represents an improvement over the Lipofectin reagent in respect to transfection of ODNs. Please follow the manufacturers guidelines for use of this product. Product sheets are available via their website. Other transfection reagents we have had previous success with include FuGene 6 from Roche Diagnostics and Superfect Transfection Reagent from Qiagen. Obviously certain cell types are more susceptible to transfection than others and certain liposome "carriers" perform better with certain cell types. Therefore an amount of trial and error may be required to optimize transfection under your specific conditions. It is therefore helpful to have a way of measuring the kinetics and efficiency of transfection of your ODN decoy. One way of doing this is to use either biotin or fluorescently labeled ODN decoys. After incubation of ODN decoys with your cells you can assess transfection efficiency by fluorescent microscopy or biotin detection. With successful transfection you should expect to see a strong nuclear signal with weaker but noticeable signal in the cytoplasm in 60-90% of your cells. We have noticed that the transfection efficiency of ODN decoys and indeed ODNs in general (for example antisense ODNs) is much more sensitive to cell density than that of plasmid DNA. Therefore we recommend that a standard seeding protocol be maintained from experiment to experiment and that cell density be varied, if required, to optimize transfection efficiency. B. The time cells are incubated with ODN decoy. The time of incubation of cells with ODN decoys is critical. While there is no standard time of incubation due to the many other variables that can affect the incubation time required (including but not limited to ODN decoy concentration, cell type and transfection reagent used) an incubation time of 8hrs (minimum) to 24-28 hrs (maximum without re-addition of ODN decoy) is suggested. Significant ODN decoy degradation has been reported to occur after incubation periods of longer than 24 hrs. Obviously frequent re-addition of ODN decoy could be used to provide continuous blockade of transcription factor functionality beyond 24 hrs if required. C. The concentration of ODN decoy used. Within the recent literature ODN decoy concentrations of up to 5mM appear to be well tolerated and highly effective in most cell types with little or no observable effect on cell viability. With the newer transfection reagents (such as OligofectAMINE) a final ODN decoy concentration within the range of 0.1-2µM will be sufficient to block transcription factor activity without inducing non-specific cellular toxicity. Controls. To confirm that the effects of the ODN decoy are due to a consensus sequence-specific inhibition of transcription factor functionality rather than a non-specific effect of the ODN decoy on cell viability or functioning we recommend using our matching mutant ODN decoys as controls in each experiment. Mutant decoys have the same flanking sequences but contain a disrupted consensus sequence that does not bind transcription factor. References Morishita, R., Higaki, J., Tomita N. and Ogihara T. (1998) Application of transcription factor "decoy" strategy as means of gene therapy and study of gene expression in cardiovascular disease. Circ Res 82, 1023-1028. Mann, M.J. and Dzau, V.J. (2000) Therapeutic applications of transcription factor decoy oligonucleotides. J. Clin. Invest. 106, 1071-1075. Products | Accounts | FAQ | Contact | Search | Home Terms of Use | Privacy Policy | Shopping Basket | Quotes | CheckoutCopyright © 2000-2007 GeneDetect.com Limited
Wiki User
∙ 15y ago
Add your answer:
Earn +20 pts
How do you locate genes on the chromosomes of humans?
Traditionally, radiolabeled oligonucleotides were used to locate genes on chromosomes. In this method, oligonucleotide sequences that are complimentary to a section of the gene being located are labelled with a radioactive isotope - P32. Following annealing, the gene location is detected through auto-radiography. These days, fluorescent labels are used. These labels are more versatile and easy to detect. They also overcome the risks of using radioactivity.
What is MLPA?
MLPA introductionMLPA (Multiplex Ligation-dependent Probe Amplification) is a multiplex PCR method detecting abnormal copy numbers of up to 50 different genomic DNA or RNA sequences, which is able to distinguish sequences differing in only one nucleotide (1). The MLPA technique is easy to use and can be performed in many laboratories, as it only requires a thermocycler and capillary electrophoresis equipment. Up to 96 samples can be handled simultaneously, with results being available within 24 hours. Although for most hereditary conditions, (partial) gene deletions or duplications account for less than 10 % of all disease-causing mutations, for many other disorders this is 10 to 30 % (2-8) or even higher still (9, 10). The inclusion of MLPA in clinical settings can therefore significantly increase the detection rate of many genetic disorders.Advantages of MLPAUsing MLPA for copy number detection offers many advantages over other techniques. First of all, methods which were primarily developed for detecting point mutations, such as sequencing and DHPLC, generally fail to detect copy numbers changes. Southern blot analysis, on the other hand, will reveal many aberrations but will not always detect small deletions and is not ideal as a routine technique. Although well-characterised deletions and amplifications can be detected by PCR, the exact breakpoint site of most deletions is unknown. Furthermore, when comparing MLPA to FISH, MLPA not only has the advantage of being a multiplex technique, but also one in which very small (50-70 NT) sequences are targeted, enabling MLPA to identify the frequent, single gene aberrations which are too small to be detected by FISH. Moreover, MLPA can be used on purified DNA. Finally, as compared to array CGH, MLPA is a low cost and technically uncomplicated method. Although MLPA is not suitable for genome-wide research screening, it is a good alternative to array-based techniques for many routine applications. The over 300 probe sets now commercially available are dedicated to applications ranging from the relatively common (Duchenne, DiGeorge syndrome, SMA) to the very rare (hereditary pancreatitis, Antithrombin deficiency, Birt-Hogg-Dube syndrome).MLPA reactionTypical for MLPA is that it is not target sequences that are amplified, but MLPA probes that hybridise to the target sequence. In contrast to a standard multiplex PCR, a single pair PCR primers is used for MLPA amplification. The resulting amplification products of a SALSA MLPA kits range between 130 and 480 NT in length and can be analysed by capillary electrophoresis. Comparing the peak pattern obtained to that of reference samples indicates which sequences show aberrant copy numbers.The MLPA reaction can be divided in five major steps: 1) DNA denaturation and hybridisation of MLPA probes; 2) ligation reaction; 3) PCR reaction; 4) separation of amplification products by electrophoresis; and 5) data analysis. During the first step, the DNA is denatured and incubated overnight with a mixture of MLPA probes. MLPA probes consist of two separate oligonucleotides, each containing one of the PCR primer sequences. The two probe oligonucleotides hybridise to immediately adjacent target sequences. Only when the two probe oligonucleotides are both hybridised to their adjacent targets can they be ligated during the ligation reaction. Because only ligated probes will be exponentially amplified during the subsequent PCR reaction, the number of probe ligation products is a measure for the number of target sequences in the sample. The amplification products are separated using capillary electrophoresis. Probe oligonucleotides that are not ligated only contain one primer sequence. As a consequence, they cannot be amplified exponentially and will not generate a signal. The removal of unbound probes is therefore unnecessary in MLPA and makes the MLPA method easy to perform.
When Chargaff separated the parts of a sample DNA what did he find out about the matching bases?
Because of the asymmetry in pyrimidine and purine use in coding sequences, the strand with the greater coding content will tend to have the greater number of purine bases (Szybalski's rule). Because the number of purine bases will to a very good approximation equal the number of their complementary pyrimidines within the same strand and because the coding sequences occupy 80-90% of the strand, there appears to be a selective pressure on the third base to minimize the number of purine bases in the strand with the greater coding content and that this pressure is proportional to the mismatch in the length of the coding sequences between the two strands. The origin of the deviation from Chargaff's rule in the organelles has been suggested to be a consequence of the mechanism of replication. During replication the DNA strands separate. In single stranded DNA, cytosine spontaneously slowly deaminates to adenosine (a C to A transversion). The longer the strands are separated the greater the quantity of deamination. For reasons that are not yet clear the strands tend to exist longer in single form in mitochondria than in chromsomal DNA. This process tends to yield one strand that is enriched in guanine (G) and thymine (T) with its complement enriched in cytosine (C) and adenosine (A), and this process may have given rise to the deviations found in the mitochondria. Chargaff's second rule appears to be the consequence of a more complex parity rule: within a single strand of DNA any oligonucleotide is present in equal numbers to its reverse complementary nucleotide. Because of the computational requirements this has not been verified in all genomes for all oligonucleotides. It has been verified for triplet oligonucleotides for a large data set. Albrecht-Buehler has suggested that this rule is the consequence of genomes evolving by a process of inversion and transposition. This process does not appear to have acted on the mitochondrial genomes. Chargaff's second parity rule appears to be extended from the nucleotide-level to populations of codon triplets, in the case of whole single-stranded Human genome DNA A kind of "codon-level second Chargaff's parity rule" is proposed as follows: Codon populations where 1st base position is T are identical to codon populations where 3rd base position is A: « % codons Twx ~ % codons yzA » (where Twx and yzA are mirror codons i.e TCG and CGA). Codon populations where 1st base position is C are identical to codon populations where 3rd base position is G: « % codons Cwx ~ % codons yzG » (where Cwx and yzG are mirror codons i.e CTA and TAG). Codon populations where 2nd base position is T are identical to codon populations where 2nd base position is A: « % codons wTx ~ % codons yAz » (where wTx and yAz are mirror codons i.e CTG and CAG). Codon populations where 2nd base position is C are identical to codon populations where 2nd base position is G: « % codons wCx ~ % codons yGz » (where wCx and yGz are mirror codons i.e TCT and AGA). Codon populations where 3rd base position is T are identical to codon populations where 1st base position is A: « % codons wxT ~ % codons Ayz » (where wxT and Ayz are mirror codons i.e CTT and AAG). Codon populations where 3rd base position is C are identical to codon populations where 1st base position is G: « % codons wxC ~ % codons Gyz » (where wxC and Gyz are mirror codons i.e GGC and GCC).
Is a butterfly masculine or feminine?
There are several ways to determine butterfly gender:(1) the way they look.(2) the way they act.(3) if they lay eggs.(4) autopsy. Males are usually:A) smaller because they have less weight (eggs) to carry, they need a greater strength to weight ratio (less wing area to muscle size) for better agility.B) have scent scales to announced their gender to a female. (also seen on the forewing of the male buckwheat copper at the beginning and end of the video, he's dusting the female's antennae with his scent) Natural selection favors healthier, less worn males with more scent scales.C) more brightly colored for.a: A poison warning.b: A flashy decoy so predators will chase the agile one, while the heavier and less obtrusive female hides.c: A territorial advertisement to other butterflies. D) have more pointed forewings for flight aerodynamics, built for speed, chase & evasion.E) have skinnier abdomens: no eggs. THERE ARE MORE WAYS THAN JUST THIS, VISIT THE "RELATED" LINK
Why Viral infection cannot be cured easily?
They can't, only white blood cells can deal with the problem because they're perfect for the job, although they may take time. Stuff like [anti-] medicines can't be used, because virus reproduce INSIDE cells, and action can't be taken unless you're sure you want your body cells to be damaged and destroyed. Bacteria CAN be cured and killed, because they DON'T reproduce inside cells
What has the author Stanley T Crooke written?
Stanley T. Crooke has written: 'Therapeutic applications of oligonucleotides' -- subject(s): Therapeutic use, Oligonucleotides 'Antisense Research and Application' 'Antisense Drug Technology'
Plural spelling for the word decoy?
The plural of decoy is decoys.
Can Someone give you a sentence with decoy in it?
Decoy the enemy so that the women and children can escape. The decoy was painted to resemble a swan.
What is recombinant PCR?
Oligonucleotides are generated and annealed to each other. Polymerase then fills in the gaps.
When was Decoy - album - created?
Decoy - album - was created in 1984-06.
Can you use the word decoy in a sentence?
A carved, painted duck may be used as a decoy by hunters to lure real ducks for the hunters to shoot. Some evangelists may use as a decoy, a religious pamphlet that looks like paper money on one side, but has a religious message on the other side.
Can you recruit a decoy?
No. A decoy is created by a Pokemon to save it from harm, it is not a Pokemon itself.
How do use decoy in sentence?
I need help finding a sentence with decoy in it helppp
When was The Decoy Bride created?
The Decoy Bride was created on 2011-05-14.
When was A Coy Decoy created?
A Coy Decoy was created on 1941-06-07.
What is the value of a JW Reynolds folding duck decoy?
Hi, What is the species of the decoy? Mike
What has the author Peter Ian Tattersall written?
Peter Ian Tattersall has written: 'Synthesis of novel oligonucleotides'
