Recombinase-Mediated Cassette Exchange (RMCE) and Integrase Swappable in vivo Targeting Element (InSITE)
Genetic switches that can turn on expression of genes of interest in specific tissues or time frames have been used in dissecting biological processes. For example, the yeast-derived transcription factor Gal4 and its upstream activating sequence (UAS) are widely adopted in Drosophila for directing gene expressions.
RMCE was designed to replace existing gene control cassette such as UAS-Gal4 with others through the functions of recombinases, such as Cre, Flp, ?C31. Multiple recombination systems were recently combined to form a flexible enhancer trap, InSITE, for exchanging Gal4 with any other sequence (1). The purpose is to use different existing fly lines that express recombinases in various types of cells.
Related to this topic, UAS-Gal4 was combined with “intra-cassette” recombination for selecting one of the three fluorescent proteins (FPs) in tandem in order to label individual neurons in a setup called “Brainbow”, which was first created in mouse, and recently in flies (2). We expect that InSITE and Drosophila Brainbow would be put together for even broader use of different fly lines and in more cell lineage studies.
One technical barrier for effective use of these systems is the lack of high quality FPs that could provide better brightness and narrower emission spectrum than those currently available. For this reason, the above referenced fly Brainbow research relied heavily on using antibodies against tags fused to the FPs, rendering live cell imaging unobtainable in most cases.
A number of the new FPs in Allele Biotech’s pipeline will be introduced to the field soon, such as the Lancelet YFP, that is ~10X brighter than EGFP, with a very narrow emission bandwidth.
(1) Gahl et Al. 2011, http://www.nature.com/nmeth/journal/v8/n3/full/nmeth.1561.html
(2) Hampel et al. 2011, http://www.nature.com/nmeth/journal/v8/n3/full/nmeth.1566.html
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Promotion of the week 022811-030611:
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By Jiwu Wang
According to the Skin Cancer Foundation, skin cancer is the most common type of cancer in the US. Although the skin might seem to be an easy target for gene therapy or RNAi mediated functional corrections, the outer keratinized epithelial cells forms a formidable barrier to delivery of genetic material. The epidermis undergoes rapid turnover, a fact that further complicates gene therapy because gene transfer to skin stem cells would be required for sustained effects.
Before skin gene therapy can be discussed with any practical meaning, a physiologically relevant in vivo model for studying gene function in the context of tumorigenesis and epithelial biology must be established. Studies of gene functions in skin homeostasis in mouse models were mostly performed by labor-intensive knockout methods. Recently, at least two publications have shown that by using ultrasound-guided injection of lentiviruses into amniotic fluids, transgene or shRNA can be efficiently and specifically delivered to epidermis, including skin stem cells, creating a very attractive model for functional studies and therapeutic tests.
Localized injection of high titer lentiviral vectors has been widely used for studying genes in brain development and a few other areas. Instead of injection into animal tissues, Endo et al. injected tiny volume (nl) of high titer lentivirus (10e10 TU/ml) into amniotic cavities within a defined window of embryogenesis . By following fluorescent protein markers (CFP, GFP, YFP, RFP), both Endo et al. and researchers from Elaine Fuchs group demonstrated high efficiency and specificity of delivery to epithelial cells, commonly resulting in multiple genomic insertions of the viral genome.
RNAi against alfa1-catenin was used by Beronja and colleagues as an example to show that loss-of-function analysis can be done rather easily using shRNA/FP bearing lentivirus . nlCre was also delivered to embryos with loxP-flanked transgenes vs wildtype for conditional knockout studies. These new findings should open doors to various experiments and therapies concerning the health of the skin.
1. Endo, M., P.W. Zoltick, W.H. Peranteau, A. Radu, N. Muvarak, M. Ito, Z. Yang, G. Cotsarelis, and A.W. Flake, Efficient in vivo targeting of epidermal stem cells by early gestational intraamniotic injection of lentiviral vector driven by the keratin 5 promoter. Mol Ther, 2008. 16(1): p. 131-7.
2. Beronja, S., G. Livshits, S. Williams, and E. Fuchs, Rapid functional dissection of genetic networks via tissue-specific transduction and RNAi in mouse embryos. Nat Med. 16(7): p. 821-7.
New Product of the Week
080210-080810: mTFP1-Mitochondria-neoR plasmid, a new drug-resistant version of Allele’s organelle markers
Promotion of the Week
080210-080810: Retroviruses expressing OSKM or OSNL set of iPS factors at $500 for order placed this week only for all Allele Facebook fans, others with code iPS0808 mentioned in order.
It seems like every week Allele Biotech has a blog entry introducing a new product, or some cutting edge research. What is often overlooked is the driving force toward this innovation, and that force is you, the customer. As a company we work to make products that address our customer needs while maintaining competitive pricing.
In order to continue these practices we rely on customer feedback. What differentiates Allele Biotech from other companies is how we react to customer input, we use it to innovate. A great example of this is Allele Mouse Tail Lysis Buffer, a product we created at the request of a customer. This customer wanted an alternative to using phenol, a potentially hazardous chemical. We were able to create our buffer which works efficiently and effectively to lyse DNA safely. Today Mouse Tail Lysis Buffer is one of our top selling reagents, but we still love to hear back from our customers about it. Other examples of customer initiated products are our Cre Lentivirus, and our Protein extraction buffer. The Cre Lentivirus was requested to give the end user a high titer virus stock for applying Cre recombinase to their MEFS. Protein Extraction Buffer was created at the behest of a customer who wanted a low cost, but high quality alternative to B-Per.
So next time you are in lab and realize you need something, or can improve upon a product send us an email. If you are already using something from Allele let us know if the protocol is unclear, or if it’s too long. Tell us if it was an easy product to use, or if it was a tedious process that can be improved upon. We want to know that our products are working for you, and that you are satisfied with your purchase from Allele Biotech. With this back and forth between company and customer we can build the ideal client relationship.
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Putting 4 iPS factors on one lentiviral vector, separated by 2A peptides, has appeared to be more efficient in generating iPS cells than having all 4 factors on individual viruses, at least in a number of cases. Stem cell-like colonies start to appear in about 2 weeks using Allele Biotech’s 4-in-1 lentivirus. In addition to the concerted effects from Oct3/4, Sox2, c-Myc, Klf4, it is also believed that the coordinated silencing of these factors after reprogramming help forming iPS colonies.
The 4-in-1 lentivirus from Allele Biotech contains loxP sites that can be used to remove the 4 cDNAs if so desired. For convenience, a new product kit is offered starting this week to include lenti-nCre in a kit with the 4-in-1 iPS viral products.
New Product of the Week 04-11-10 to 04-18-10: 4-In-One-Vector: Human OSKM Lentiviral Paticles (Oct3/4, Sox2, Klf4 and c-Myc) and Cre Lentiviral Particle kits, Cat # ABP-SC-LVI4IN1C1 or ABP-SC-LVI4IN1C5
Promotion of the Week 04-11-10 to 04-18-10: Single vial 4-in-1 is offered only this week. This product has been well established and validated, one of the reasons smaller packages are not normally offered. As a matter of fact, every batch of the 4-in-1 iPS lentivirus has been sold out.
The bacterial Cre recombinase targets a specific DNA sequence called loxP and deletes a segment of DNA flanked by loxP sequences. This system is often used in the generation of knockout and conditional knockout animals.
The knockout of specific genes leading to embryonic lethal phenotype will not yield adult animals. Cre-lox recombination provides a means to knockout the specific genes in adult mice, or to introduce a knockout phenotype in specific tissues (conditional knockout) using tissue-specific promoter driven Cre or an inducible Cre.
The cutting by Cre at the loxP sites and rejoining by ligase is an efficient process. During this process, inverted loxP sites will result in an inversion, whereas direct repeat will cause a deletion. Cre/lox recombination is a one-way reaction so there is no need for continued Cre expression. Therefore, Cre can be introduced by adenovirus or lenti/retrovirus. Here is an example of using adnovirus-Cre in one lab: for MEF, on a 70% confluent P10 cm plate (probably 2-2.5 million cells), use 6ul of 1.1×10^12 adenovirus-Cre, which will give 80% infection; or use 10ul of 1.1×10^12 adenovirus-Cre to get 90% infection, with GFP as marker and analyzed by FACS.
Adenovirus could post a toxicity problem when used at very high titers to reach high percentage of transduction. An alternative is to use only lentivirus-Cre, at only about 1-2 ul and still obtain >80% infection. However, a silencing event needs to occur before the expression of Cre from lentivirus is shut off. The timing and degree of silencing is not controlled in such experiments. Continued expression of Cre should not influence most experiments.
To be certain that the Cre enzyme can be successfully delivered into the nucleus for conditional knockout to occur, the bacterial Cre gene needs to be engineered to contain a nuclear localization (nl) signal of eukaryotic cells. The function of the nuclear-localized Cre (nlCre) can be tested using a loxP-nuclear localized lacZ (nlacZ) reporter cell line, which can be used to monitor the function of the nlCre recombinase.
New Product of the Week 03-01-10 to 03-07-10: Lentivirus-Cre ABP-RP-TLCCreS and ABP-RP-TLCCreL, link: http://www.allelebiotech.com/shopcart/index.php?c=219&sc=188.
Promotion of the Week 03-01-10 to 03-07-10: for a limited time, get lab agarose at ~1/5 of the big company’s price. http://www.facebook.com/#!/pages/San-Diego-CA/Allele-Biotechnology-and-Pharmaceuticals-Inc/78331924957
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