The ballot is in—among the “usual suspect” hot topics, iPS takes the top honor and most entries; Camelid antibodies, although not really presented as a typical AlleleBlog in 2011, made it to the top 3. shRNA cloning and RNAi screening are still on a lot of people’s minds, so it seems.
Method: total visits to each blog since our new webpage was launched in July was counted.
1) Fusion of the Transcription Domain to iPS Factors Radically Enhances Reprogramming
http://blog.allelebiotech.com/2011/10/fusion-of-the-transcription-domain-to-ips-factors-radically-enhances-reprogramming/
2) Methods of iPSC Generation Update
http://blog.allelebiotech.com/2011/08/methods-of-ipsc-generation-update/
3) About 50 Papers Cited the Use of GFP-Trap Camelid Antibody So Far in 2011
http://blog.allelebiotech.com/2011/09/about-50-papers-cited-the-use-of-gfp-trap-camelid-antibody-so-far-in-2011/
4) Big Potential in Using Protozoans for Producing Mammalian Proteins
http://blog.allelebiotech.com/2011/09/big-potential-in-using-protozoans-for-producing-mammalian-proteins/
5) How do you make shRNA-expressing viruses for function screening?
http://blog.allelebiotech.com/2011/11/how-do-you-make-shrna-expressing-viruses-for-function-screening/
6) Creating ground-state human iPSCs
http://blog.allelebiotech.com/2011/10/creating-ground-state-human-ipsc/
7) Recombinase-Mediated Cassette Exchange (RMCE) and Integrase Swappable in vivo Targeting Element (InSITE)
http://blog.allelebiotech.com/2011/03/recombinase-mediated-cassette-exchange-rmce-and-integrase-swappable-in-vivo-targeting-element-insite/
8) Development of Cell Lines from iPSCs for Bioassays
http://blog.allelebiotech.com/2011/11/development-of-cell-lines-from-ipscs-for-bioassays/
9) Choosing siRNA, shRNA, and miRNA for Gene Silencing
blog.allelebiotech.com/2010/02/choosing-sirna-shrna-and-mirna-for-gene-silencing/
10) Allele Biotech’s Box Swap Program
http://blog.allelebiotech.com/2009/07/allele-biotechs-box-swap-program/
Have a successful 2012!
From AlleleBlog: http://blog.allelebiotech.com/2011/12/top-10-list-of-most-viewed-alleleblogs-in-2011/
Showing posts with label RNAi screening. Show all posts
Showing posts with label RNAi screening. Show all posts
Saturday, December 31, 2011
Wednesday, November 16, 2011
How do you make shRNA-expressing viruses for function screening?
Allele Weekly Blog: http://blog.allelebiotech.com/2011/11/how-do-you-make-shrna-expressing-viruses-for-function-screening/
Most people use standard cloning procedures when trying to insert shRNA templates into lentiviral vectors, i.e. anneal a pair of long oligos with sticky ends and ligate the dsDNA into a linearized plasmid with compatible overhangs. However, since typical lentiviral vector plasmids have terminal repeats and are relatively large, when ligated to hairpin sequence-containing shRNA templates, recombination often occurs inside bacteria that results in smaller plasmids. This problem is common for cloning shRNA or other unstable DNA pieces into viral vectors. This cloning issue is further compounded by the fact that it is difficult to sequence any shRNA template region because the hairpin may block the progress of the DNA polymerase used in sequencing, sometimes requiring several repeats under different sequencing conditions, incurring high costs charged by sequencing service providers.
To deal with these aspects of the cloning difficulties, particularly for the purpose of increasing cloning efficiency RNAi-based screening, we compared three different strategies
First, we built a smaller shRNA cloning vector to clone and sequence shRNA templates prior to transferring to lentiviral vectors. This smaller vector does not have a severe recombination problem and is easier to sequence in the hairpin-containing region. After an initial round of cloning with this new vector, we further improved it by inserting an XbaI and a NheI site between the BamHI and SpeI insertion sites, so that any plasmid preparations can be screened for recombinants by a simple XbaI or NheI digest before sequencing. After cloning into this intermediate vector, the shRNA expression cassette can be transferred into the lentivirus vectors with some flanking viral sequences so that the insert size will be around 1kb.
Second, we developed a novel DNA preparation procedure after realizing that DNA damage during miniprep of vector plasmids and gel purification of vector fragment increased recombination of these constructs, which were already less stable than usual due to hairpin structures. This procedure of DNA preparation avoids UV or guanidium exposure, which can cause nicks on double-stranded DNA and facilitate recombination. This new procedure relies on purifying DNA through surface-binding to regular reaction tubes treated with a proprietary reagent (SurfaceBind Purification). The process simply requires adding a proprietary, guanidium-free binding buffer to the DNA, which has been processed in a specially coated tube (eppendorf or thin-wall PCR tube), and purifying directly in the same tube. Vectors prepared this way indeed provide more colony counts and a higher percentage of correct constructs as shown by our test runs. The procedure also requires less time and the purified DNA can be dissolved in volumes as small as a few microliters.
Third, to enable truly high throughput shRNA screening (i.e. looking for effective RNAi reagents), we further tested and adapted a ligationless cloning protocol that can be handled by a liquid handler almost entirely. In order to increase throughput, we designed a drastically different procedure that could bypass ligation and sequencing altogether before functional tests. Briefly, DNA molecules that would provide enhanced recombination were created by one round of PCR, purified directly in the surface bind PCR reaction tubes (any template DNA would be removed with DpnI enzyme that cuts non-PCR DNA), pooled, and transformed in bacteria directly. DNA plasmids from transformed bacteria can be used for lentivirus packaging, bypassing sequencing at the initial screening stage, and choose single colonies for sequencing only after a shRNA sequence shows promise in functional assays. This is based on the fact that such cloning rarely has any background colonies, and that among all oligos (if using the correct grade of oligos from validated suppliers) inserted this way, a good portion encodes the correct sequence.
New Products of the week: 100x 15mm EcoCulture Vented Dishes for better stem cell attachment and less plastic waste to the environment, APB-CS-114TC.
Promotion of the week: Buy 1 Stealth Express IPS Induction PCR Template Set, get 1 SurfaceBind RNA Purification Kit free. Use code FreePureRNA.
Friday, March 19, 2010
Fluorescent Protein-Based Assay Development
This blog will be the template of Allele's new cell based assay service landing page. http://www.allelebiotech.com/allele3/index.php
Overview:
Originally cloned from the jellyfish Aequorea victoria and subsequently from many other marine organisms, fluorescent proteins (FPs) spanning the entire visual spectrum have become some of the most widely used genetically encoded tags. Unlike traditional labeling methods, FPs may be used to specifically label virtually any protein of interest in a living cell with minimal perturbation to its endogenous function. 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. Once expressed, FPs are easily detected with standard fluorescence microscopy equipment.
Factors that should be taken into account when designing an FP-based imaging experiment include the desired wavelength(s) for detection, the pH environment of the tagged protein, the total required imaging time, and the expression level or dynamic range required for detection of promoter activity or tagged protein. Individual FPs currently available to the research community vary considerably in their photostability, pH sensitivity, and overall brightness, and so FPs must be chosen with care to maximize the likelihood of success in a particular experimental context.
FPs as fusion tags:
Use of FPs as fusion tags allows visualization of the dynamic localization of the tagged protein in living cells. For such applications, the cDNA of a protein of interest is attached in-frame to the coding sequence for the desired FP, and both are put under the control of a promoter appropriate to the experimental context (typically CMV for high-level expression, though other promoters may be desirable if overexpression of your protein of interest is suspected of producing artifacts). The most basic uses for fluorescent protein fusions include tracking of specific organelles (fusions to short organelle targeting signals) or cytoskeletal structures (fusions to actin or tubulin, for example). More advanced uses include tracking receptors or exported proteins. In most cases, it is critical that the FP used for fusion tagging be fully monomeric, as any interaction between fusion tags is likely to produce artifacts, some of which may be hard to recognize in the absence of other controls. While in most cases FP fusions do not interfere with normal protein function, whenever possible, FP fusion proteins should be validated by immunostaining the corresponding endogenous protein in non-transfected cells and verifying similar patterns of localization.
FPs as expression reporters:
FPs are highly useful as quantitative expression reporters. By driving the expression of an FP gene by a specific promoter of interest, it is possible to produce an optical readout of promoter activity. Use of the brightest possible FP ensures the best dynamic range for such an experiment. Because dynamic localization is not generally an issue for expression reporter applications, it is possible to use non-monomeric FPs for this purpose, opening up additional possibilities for multiple wavelength imaging. In order to obtain more reliable quantitative data and to correct for likely variations between individual cells in expression reporter experiments, the use of two spectrally distinct (e.g. green and red) FPs is advisable. By driving expression of one FP with a constitutive promoter and a second FP with the promoter of interest, the ratio of the two signals provides a quantitative readout of relative activity. Averaged over many cells, this technique should provide statistical power necessary for quality expression level experiments. Because FPs normally have a very slow turnover rate in mammalian cells, it may be desirable to add a degradation tag to your FP to enhance temporal resolution when measuring highly dynamic promoter activity.
Overview:
Originally cloned from the jellyfish Aequorea victoria and subsequently from many other marine organisms, fluorescent proteins (FPs) spanning the entire visual spectrum have become some of the most widely used genetically encoded tags. Unlike traditional labeling methods, FPs may be used to specifically label virtually any protein of interest in a living cell with minimal perturbation to its endogenous function. 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. Once expressed, FPs are easily detected with standard fluorescence microscopy equipment.
Factors that should be taken into account when designing an FP-based imaging experiment include the desired wavelength(s) for detection, the pH environment of the tagged protein, the total required imaging time, and the expression level or dynamic range required for detection of promoter activity or tagged protein. Individual FPs currently available to the research community vary considerably in their photostability, pH sensitivity, and overall brightness, and so FPs must be chosen with care to maximize the likelihood of success in a particular experimental context.
FPs as fusion tags:
Use of FPs as fusion tags allows visualization of the dynamic localization of the tagged protein in living cells. For such applications, the cDNA of a protein of interest is attached in-frame to the coding sequence for the desired FP, and both are put under the control of a promoter appropriate to the experimental context (typically CMV for high-level expression, though other promoters may be desirable if overexpression of your protein of interest is suspected of producing artifacts). The most basic uses for fluorescent protein fusions include tracking of specific organelles (fusions to short organelle targeting signals) or cytoskeletal structures (fusions to actin or tubulin, for example). More advanced uses include tracking receptors or exported proteins. In most cases, it is critical that the FP used for fusion tagging be fully monomeric, as any interaction between fusion tags is likely to produce artifacts, some of which may be hard to recognize in the absence of other controls. While in most cases FP fusions do not interfere with normal protein function, whenever possible, FP fusion proteins should be validated by immunostaining the corresponding endogenous protein in non-transfected cells and verifying similar patterns of localization.
FPs as expression reporters:
FPs are highly useful as quantitative expression reporters. By driving the expression of an FP gene by a specific promoter of interest, it is possible to produce an optical readout of promoter activity. Use of the brightest possible FP ensures the best dynamic range for such an experiment. Because dynamic localization is not generally an issue for expression reporter applications, it is possible to use non-monomeric FPs for this purpose, opening up additional possibilities for multiple wavelength imaging. In order to obtain more reliable quantitative data and to correct for likely variations between individual cells in expression reporter experiments, the use of two spectrally distinct (e.g. green and red) FPs is advisable. By driving expression of one FP with a constitutive promoter and a second FP with the promoter of interest, the ratio of the two signals provides a quantitative readout of relative activity. Averaged over many cells, this technique should provide statistical power necessary for quality expression level experiments. Because FPs normally have a very slow turnover rate in mammalian cells, it may be desirable to add a degradation tag to your FP to enhance temporal resolution when measuring highly dynamic promoter activity.
New Product of the Week 03-15-10 to 03-21-10: Oct4-Sox2 2-in-1 lentivirus ABP-SC-LVI2in1 for effective iPS generation link: http://www.allelebiotech.com/shopcart/index.php?c=132&sc=122.
Promotion of the Week 03-15-10 to 03-21-10: 5% off plate oligos at all scales! www.allelebiotech.com/allele3/Oligo_96Plate.php We are doing our “window promotion” again, during a hour-long window, get any Allele’s High efficiency competent cells at 30% regular price, the time will be announced tomorrow on our Facebook page.
Overview:
Originally cloned from the jellyfish Aequorea victoria and subsequently from many other marine organisms, fluorescent proteins (FPs) spanning the entire visual spectrum have become some of the most widely used genetically encoded tags. Unlike traditional labeling methods, FPs may be used to specifically label virtually any protein of interest in a living cell with minimal perturbation to its endogenous function. 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. Once expressed, FPs are easily detected with standard fluorescence microscopy equipment.
Factors that should be taken into account when designing an FP-based imaging experiment include the desired wavelength(s) for detection, the pH environment of the tagged protein, the total required imaging time, and the expression level or dynamic range required for detection of promoter activity or tagged protein. Individual FPs currently available to the research community vary considerably in their photostability, pH sensitivity, and overall brightness, and so FPs must be chosen with care to maximize the likelihood of success in a particular experimental context.
FPs as fusion tags:
Use of FPs as fusion tags allows visualization of the dynamic localization of the tagged protein in living cells. For such applications, the cDNA of a protein of interest is attached in-frame to the coding sequence for the desired FP, and both are put under the control of a promoter appropriate to the experimental context (typically CMV for high-level expression, though other promoters may be desirable if overexpression of your protein of interest is suspected of producing artifacts). The most basic uses for fluorescent protein fusions include tracking of specific organelles (fusions to short organelle targeting signals) or cytoskeletal structures (fusions to actin or tubulin, for example). More advanced uses include tracking receptors or exported proteins. In most cases, it is critical that the FP used for fusion tagging be fully monomeric, as any interaction between fusion tags is likely to produce artifacts, some of which may be hard to recognize in the absence of other controls. While in most cases FP fusions do not interfere with normal protein function, whenever possible, FP fusion proteins should be validated by immunostaining the corresponding endogenous protein in non-transfected cells and verifying similar patterns of localization.
FPs as expression reporters:
FPs are highly useful as quantitative expression reporters. By driving the expression of an FP gene by a specific promoter of interest, it is possible to produce an optical readout of promoter activity. Use of the brightest possible FP ensures the best dynamic range for such an experiment. Because dynamic localization is not generally an issue for expression reporter applications, it is possible to use non-monomeric FPs for this purpose, opening up additional possibilities for multiple wavelength imaging. In order to obtain more reliable quantitative data and to correct for likely variations between individual cells in expression reporter experiments, the use of two spectrally distinct (e.g. green and red) FPs is advisable. By driving expression of one FP with a constitutive promoter and a second FP with the promoter of interest, the ratio of the two signals provides a quantitative readout of relative activity. Averaged over many cells, this technique should provide statistical power necessary for quality expression level experiments. Because FPs normally have a very slow turnover rate in mammalian cells, it may be desirable to add a degradation tag to your FP to enhance temporal resolution when measuring highly dynamic promoter activity.
Overview:
Originally cloned from the jellyfish Aequorea victoria and subsequently from many other marine organisms, fluorescent proteins (FPs) spanning the entire visual spectrum have become some of the most widely used genetically encoded tags. Unlike traditional labeling methods, FPs may be used to specifically label virtually any protein of interest in a living cell with minimal perturbation to its endogenous function. 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. Once expressed, FPs are easily detected with standard fluorescence microscopy equipment.
Factors that should be taken into account when designing an FP-based imaging experiment include the desired wavelength(s) for detection, the pH environment of the tagged protein, the total required imaging time, and the expression level or dynamic range required for detection of promoter activity or tagged protein. Individual FPs currently available to the research community vary considerably in their photostability, pH sensitivity, and overall brightness, and so FPs must be chosen with care to maximize the likelihood of success in a particular experimental context.
FPs as fusion tags:
Use of FPs as fusion tags allows visualization of the dynamic localization of the tagged protein in living cells. For such applications, the cDNA of a protein of interest is attached in-frame to the coding sequence for the desired FP, and both are put under the control of a promoter appropriate to the experimental context (typically CMV for high-level expression, though other promoters may be desirable if overexpression of your protein of interest is suspected of producing artifacts). The most basic uses for fluorescent protein fusions include tracking of specific organelles (fusions to short organelle targeting signals) or cytoskeletal structures (fusions to actin or tubulin, for example). More advanced uses include tracking receptors or exported proteins. In most cases, it is critical that the FP used for fusion tagging be fully monomeric, as any interaction between fusion tags is likely to produce artifacts, some of which may be hard to recognize in the absence of other controls. While in most cases FP fusions do not interfere with normal protein function, whenever possible, FP fusion proteins should be validated by immunostaining the corresponding endogenous protein in non-transfected cells and verifying similar patterns of localization.
FPs as expression reporters:
FPs are highly useful as quantitative expression reporters. By driving the expression of an FP gene by a specific promoter of interest, it is possible to produce an optical readout of promoter activity. Use of the brightest possible FP ensures the best dynamic range for such an experiment. Because dynamic localization is not generally an issue for expression reporter applications, it is possible to use non-monomeric FPs for this purpose, opening up additional possibilities for multiple wavelength imaging. In order to obtain more reliable quantitative data and to correct for likely variations between individual cells in expression reporter experiments, the use of two spectrally distinct (e.g. green and red) FPs is advisable. By driving expression of one FP with a constitutive promoter and a second FP with the promoter of interest, the ratio of the two signals provides a quantitative readout of relative activity. Averaged over many cells, this technique should provide statistical power necessary for quality expression level experiments. Because FPs normally have a very slow turnover rate in mammalian cells, it may be desirable to add a degradation tag to your FP to enhance temporal resolution when measuring highly dynamic promoter activity.
New Product of the Week 03-15-10 to 03-21-10: Oct4-Sox2 2-in-1 lentivirus ABP-SC-LVI2in1 for effective iPS generation link: http://www.allelebiotech.com/shopcart/index.php?c=132&sc=122.
Promotion of the Week 03-15-10 to 03-21-10: 5% off plate oligos at all scales! www.allelebiotech.com/allele3/Oligo_96Plate.php We are doing our “window promotion” again, during a hour-long window, get any Allele’s High efficiency competent cells at 30% regular price, the time will be announced tomorrow on our Facebook page.
Tuesday, November 24, 2009
Allele Will Receive Its 3rd US Patent On RNAi
On December 1st, 2009 Allele Biotech will be granted its 3rd US patent on using RNA polymerase III (Pol III) for creating RNAi inside mammalian cells. Previously, US patent 7,294,504 was granted to Allele Biotech that covers commercial kits with DNA template components designed for expression of shRNA, miRNA, or siRNA; US patent 7,422,896 further granted claims covering broader designs of using a Pol III promoter such as a U6 promoter for RNAi, including the use of a constitutive or inducible enhancer. The current US patent 7,625,750, protects the use of above technologies in conjunction with arrays, particularly addressable, high density DNA arrays. The RNAi encoding DNA molecules, anchored to the surface through a special peptide, can be transduced via transduction peptide into target cells grown on the array surface. The DNA will then be released from the surface after the completion of DNA transfer by a membrane protease. "This array format for RNAi using the Pol III technology should have higher efficiency and controllability than soft agar embedded siRNAs for transfecting cells, with great potential in large-scale RNAi functional screening and validation. Combined with Allele Biotech’s existing lentiviral vector-based shRNA platform, the addressable RNAi arrays provide us with the best methods available for RNAi screening”, said Dr. Jiwu Wang, CEO of Allele Biotech and the inventor of the patent.
Allele Biotech provides reagent kits and custom services from using its patented technologies in the field of RNAi. The 3 patents issued to Allele Biotech within the past 2 years are so far the only US patents on the methods and compositions of using Pol III promoter for expressing dsRNA-mediated gene silencing. Allele Biotech aims to strengthen its market position by providing superior products and services while actively protecting its intellectual properties. The current strategy includes noting providers and users of existing products that apparently fall under Allele’s patent protection in order to provide reasonable sub-licensing or co-development options.
Allele Biotech is expanding its RNAi capabilities by incorporating the Pol III promoter-driven shRNA cassettes into its popular Phoenix retroviral system and the recently added lentiviral vector system. The RNAi service will also be integrated with Allele’s viral packaging service which offers the best value in terms of per viral particle cost in the market today. High throughput and high content screenings to be conducted at Allele could be further aided by the use of low mutation oligo annealing and wall-less cell array technologies by collaborating with partner companies. In addition, RNAi target design and selection are carried out with an advanced algorism and the most effective empirical rules through Allele’s RNAi services.
Allele Biotech's RNAi technologies were developed with help from the National Institutes of Health (NIH) through several grants. The research team at Allele is currently applying for another NIH project to use these technologies in synthetic lethal screening for cancer therapy. Dr. Wang said that "It is now our goal as well as responsibility to make the RNAi technologies helpful to as many researchers as possible in their pursuit of the best results from gene function studies”.
Allele Biotech provides reagent kits and custom services from using its patented technologies in the field of RNAi. The 3 patents issued to Allele Biotech within the past 2 years are so far the only US patents on the methods and compositions of using Pol III promoter for expressing dsRNA-mediated gene silencing. Allele Biotech aims to strengthen its market position by providing superior products and services while actively protecting its intellectual properties. The current strategy includes noting providers and users of existing products that apparently fall under Allele’s patent protection in order to provide reasonable sub-licensing or co-development options.
Allele Biotech is expanding its RNAi capabilities by incorporating the Pol III promoter-driven shRNA cassettes into its popular Phoenix retroviral system and the recently added lentiviral vector system. The RNAi service will also be integrated with Allele’s viral packaging service which offers the best value in terms of per viral particle cost in the market today. High throughput and high content screenings to be conducted at Allele could be further aided by the use of low mutation oligo annealing and wall-less cell array technologies by collaborating with partner companies. In addition, RNAi target design and selection are carried out with an advanced algorism and the most effective empirical rules through Allele’s RNAi services.
Allele Biotech's RNAi technologies were developed with help from the National Institutes of Health (NIH) through several grants. The research team at Allele is currently applying for another NIH project to use these technologies in synthetic lethal screening for cancer therapy. Dr. Wang said that "It is now our goal as well as responsibility to make the RNAi technologies helpful to as many researchers as possible in their pursuit of the best results from gene function studies”.
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