Fluorescent proteins
Delivery of RNAi or Cre by Ultrasound-Guided Injection of High Titer Lentiviral Vectors
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 [1]. 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 [2]. 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.
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Promotion of the Week
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Allele’s pallet of the super star fluorescent proteins
“Photoblog”–just some fun pictures from our notebooks.
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The brightest cyan, green fluorescent proteins, and the brightest ever FP in LanYFP!
Ain't they pretty?
These fluorescent proteins are representatives of the growing family or high quality, new generation FPs engineered to enable experiment previously deemed impossible.
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Cells infected with lentivirus carrying mWasabi. Lentivirus carrying LanYFP will make most cells much more brighter than this.
The mWasabi is stimulating
The brightest green fluorescent protein with excellent photostability, carried on 10e8 TU/ml high titer lentivirus.
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The LanFPs express well in bacteria.
The LanFPs express well in bacteria
Project planning is under way to test the cytotoxicity of lanFPs in different mammalian cell lines and in vivo with a focus on neurons.
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The FPs fold so strongly that they fluorescence even in SDS-PAGE.
Can you see the FP bands in the SDS PAGE?
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FPs in SDS PAGE–a closer look
Can you see them now?
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FPs in gel cassette over UV lights
Invincible FPs
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FPs in gel cassette under blue LED
Fluorescence in SDS page under blue LED
The purified FPs can be used as “real time” protein markers.
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Brightest Ever Fluorescent Protein
LanYFP, identified from lancelet (also known as amphioxus, e.g. Branchiostoma floridae), has been found to have the following properties:
Excitation 513nm
Emission 524nm
Quantum yield 0.95
Extinction coefficient 150,000
pKa ~3.5
Salt insensitive 0-500mM NaCl
LanYFP has a brightness of 143! For comparison, the brightness of the previously known brightest FPs is 95 for tdTomato, and 34 for commonly used EGFP.
Allele already has been exclusively providing the brightest cyan FP in mTFP1 (brightness of 54); and the brightest green FP in mWasabi (brightness of 56). The confirmation of LanYFP as the brightest ever FP is a major milestone of Allele’s research and development efforts in the fluorescent protein field. We are currently monomerizing LanYFP and another lancelet protein, LanRFP. Once completed, the new proteins should definitely be the FPs of choice for in vivo imaging and FRET with unprecedented utilities.
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New product of the week, recombinant mTFP1, mWasabi, LanYFP, LanRFP, $159 for 125 ug, compare price for 100ug vs 125ug in other companies’ offers, you will know that you are getting a good deal from Allele.
Lentivirus-lanYFP Give Away
Promotion of the week 03-22-10 to 03-28-10: To help researchers get familiar with pre-packaged lentivirus, we offer free high titer lentivirus carrying a truly bright and fast maturing lanYFP (lancelet FP, new exclusively from Allele). Infect virtually any mammalian cells by a single manipulation (pipeting) and watch cells turn green/yellow in about a day under microscope or on FACS. Primarily a yellow FP, lanYFP will show brighter fluorescence than EGFP even when observed using standard GFP/FITC filter set.
New product of the week 03-22-10 to 03-28-10: anti-mTFP1/mWasabi polyclonal antibodies. It is tailored-made for Alleleustrious mTFP1 and mWasabi, the brightest teal and green FPs.
Fluorescent Protein-Based Assay Development II
FPs as pH and redox sensors:
The uses of FPs extend well beyond simple expression and fusion reporters. While pH sensitivity (usually quenching of fluorescence by acidic pH) is generally considered a drawback for fusion tagging, it becomes a useful property for constructing pH sensors. FPs specifically engineered to take advantage of pH sensitivity (“pHluorins”) report pH as either a change in fluorescent intensity or a change in the ratio of excitation at two different wavelengths, and may be used to monitor processes such as endocytosis or other pH-variable processes. In such an application, the pH-sensitive FP is fused to a localization tag for the compartment of interest which experiences variable pH. This technique can be used, for example, to visualize release of neurotransmitter-containing vesicles. In addition to pH-sensitive FPs, redox-sensitive Aequorea GFP variants have been produced (roGFPs and others) which produce similar changes in fluorescence intensity or excitation ratio when exposed to differing redox conditions or reactive oxygen species.
Sensors based on circularly permuted FPs:
Because FPs have such a compact and stable beta-barrel fold with N and C termini close together, it is possible to engineer circularly permuted variants which retain their fluorescent properties. Studies on circular permutation of FPs have led to the development of several different sensors which take advantage of domains inserted into sensitive areas of the fluorescent protein backbone. The most famous of these are the GCaMP calcium sensors, in which a calmodulin domain has been inserted into a loop in GFP, yielding a sensor that reports calcium concentration as a change in fluorescent intensity. Other circularly permuted FP variants, such as cpVenus (a yellow Aequorea GFP variant), have found usefulness in improving FRET sensor dynamic range (see next section).
FRET sensors:
Fluorescence resonance energy transfer (FRET) is a quantum mechanical process that allows the transfer of excited state energy between two fluorophores when they are in close physical proximity. Because this process operates with a strong distance (1/r^6) and orientation dependence (strongest when chromophore dipoles are parallel or antiparallel), it lends itself to the construction of highly sensitive reporters of biochemical activity. In FP FRET, excited state energy from a higher-energy (shorter wavelength) “donor” fluorescent protein is transferred to a lower-energy (longer wavelength) “acceptor” FP, leading to sensitized fluorescent emission from the acceptor and reduced emission (quenching) from the donor. By linking donor and acceptor FPs with a domain which changes conformation in response to a biochemical activity of interest, this activity is reported as a change in the ratio of sensitized emission to direct-excitation emission of the acceptor (or a simple ratio of donor and acceptor emission). FRET sensors have been engineered to specifically sense a wide variety of activities, including many protein kinases, as well as small molecules such as Ca2+ and neurotransmitters. While design of a new FRET sensor generally requires a great deal of optimization and trail-and-error, this class of probe is among the most powerful tools currently available for investigating live-cell biochemistry.
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