Another potential improvement to the process would be to use bacterial expression to screen for transposon insertions that produce a fluorescent protein. This could, however, be problematic with proteins from the mammalian nervous sytem, such as ion channels, that are difficult to express in bacteria. The approach described here should speed the discovery of genetically encodable fluorescent sensors. This discovery, however, was the result of designing, building, and testing eight different tribrid fusion proteins [ 22 ].
Little is known about the mechanism whereby changes in channel conformation are converted to changes in the fluorophore, so it remains to be determined whether GFP can signal conformational changes in other kinds of proteins. Nevertheless, the use of the transposons described here should shift the work from building the constructs to devising high throughput assays for function.
Finally, random GFP tagging will facilitate the creation of potential fluorescence resonance energy transfer FRET reagents to study protein interactions in living systems. To date, a few studies have demonstrated the potential power of GFP-FRET by labeling different proteins [ 6 , 23 — 26 ] or by fusing two different fluorophores to the same protein [ 27 — 32 ].
Creating efficent donor and acceptor fusion proteins is difficult, however, because FRET only occurs when the two fluorophores are attached to surfaces that are very close to one another. The approach described here makes it possible to rapidly generate libraries of potential donor and acceptor tribrid fusion proteins that can be screened, in pairwise combinations, for function and FRET signals.
The transposons described here make it possible to rapidly generate large numbers of different GFP fusion proteins. The results show that GFP can be inserted into a wide variety of other protein domains and it will continue to fold and form a fluorophore.
The rapid and random nature of the transposition process makes it possible to generate and screen many different fusion constructs to identify those that continue to function. In the case of the two proteins tested here, roughly 1 in 6 of the fusion proteins retained their signaling function, and the random nature of the transposition process revealed permissive sites for insertion that would not have been predicted on the basis of structural or functional models of how that protein works.
This simple tool should speed the search for a wide variety of new biological probes for the study of nervous system. Molar equivalents of transposon and target plasmid 0.
Top 10 F' E. Linker sequences are in brackets. Srf I restriction digestion was then used to remove the Kan r cassette from the clones carrying in-frame insertions, thereby creating a sequence encoding a full-length fusion protein.
After digestion and re-ligation, Top 10 F' E. The colonies were re-plated the following day on ampicillin and kanamycin to verify loss of the Kan r. The fusion proteins were transiently expressed in HEK cells [ 37 ]. Images were collected from live cells 20—48 hr later on an inverted Zeiss microscope fitted with computer controlled IPLabs, Scanalytics filter wheels Ludl Electronics on the excitation and emission paths.
Forty-eight hours after transfection, cells were lysed and membrane and supernatant fractions harvested as described previously [ 8 ]. Whole-cell patch clamp recording was used to test the GluR1 fusion proteins for function in transiently transfected HEK cells as previously described [ 41 ]. All chemicals were purchased from Sigma. Drugs were applied with a rapid superfusion system made from a pulled theta capillary. Chalfie M: Green fluorescent protein. Photochem Photobiol. Nucleic Acids Res.
Association with the plasma membrane is disrupted by mutational activation and by elimination of palmitoylation sites, but not by activation mediated by receptors or AlF4. J Biol Chem. Biochem Biophys Res Commun. Nat Biotechnol. Borjigin J, Nathans J: Bovine pancreatic trypsin inhibitor-trypsin complex as a detection system for recombinant proteins. Ataka K, Pieribone VA: A genetically targetable fluorescent probe of channel gating with rapid kinetics.
Biophys J. Eur J Neurosci. Nat Struct Biol. PCR methods and applications. In addition, translational gene fusions can also provide information about subcellular localization and the temporal aspects of gene regulation. Translational fusions may appear less bright than transcriptional fusions due to the intrinsic instability of the protein fused to GFP. However, insertion of GFP intragenically can sometimes disrupt protein function or even lead to toxicity of the chimeric product.
Finally, translational reporters that exhibit subcellular localization can make cell type identification more difficult because the shape of the cell may not be visible especially for neurons.
GFP produced by these reporters is not localized which can facilitate cell type identification. The major disadvantage of smg-1 -based reporters is that they require a smg-1 mutation in the genetic background. A large number of vector backbones and reporter gene derivatives are available from the Fire Vector Kit constructed by Andrew Fire's lab. Some of the most useful vectors from the Fire Vector Kit are listed in Table 1.
Table 1. Of little consequence to the experimentalist, yet fascinating in its own right, is the autofluorescence exhibited by the gut granules and nucleoli of hypodermal cells in C. These phenomena are not a result of aberrant reporter transgene expression, but occur naturally in C. In contrast to non-GFP-related autofluorescence, GFP reporters can sometimes induce non-specific fluorescence in posterior gut cells.
Temporal gene regulation studies rely on the ability to detect gene expression fluctuations within a narrow time window. A drawback to using fluorescent proteins for such experiments is the time required for the maturation of the fluorophore.
Studies performed in E. Evidence from C. The red fluorescent proteins, DsRed and DsRed2, mature significantly slower. However, new variants of DsRed have been generated that are brigther, mature faster Shaner et al.
Sometimes expression from weak promoters may require a prolonged accumulation of protein before a fluorescent signal is detectable in vivo. In these cases, antibody staining can reveal expression of the fluorescent protein before a detectable fluorescent signal is achieved.
A number of standard coinjection markers are available to label reporter gene-containing extrachromosomal arrays in C. The most commonly used marker is rol-6 su Kramer et al. Alternative injection markers that do require specific mutants background include: pha-1 , dpy , unc-4 , lin see WormMethods section: Transformation and microinjection.
Notably, GFP reporters, such as ceh :: gfp , unc :: gfp , elt-2 :: gfp , and ttx-3 :: gfp have been used as injection markers in their own right. Reporter transgenes may not represent the complete expression pattern for a gene, but still provide testable hypotheses about the site of gene function. It has become standard to supplement these hypotheses with cell-type specific rescue experiments, mosaic analysis and antibody staining.
For early embryo studies, in situ hybridization can also be informative. The most common approach for generating reporter genes in C. A large array of vectors have been made by Andrew Fire's lab. All vectors have a backbone based on the pUC19 plasmid and contain the E. They also have convenient multiple cloning sites MCS and provide a number of useful reporter gene variants.
In addition to the Fire Vector Kit, many expression vectors are available in the C. The reader is expected to be familiar with standard molecular cloning techniques. Protocols will be focusing on specific aspects relating to C. The first step in designing GFP reporter constructs is to choose an adequate vector backbone. Many vectors are available in the Fire Kit. Presented in Figure 2 is the structure of pPD In addition, a variety of specialized vectors are listed in Table 1.
Figure 2. Description of pPD The reading frame upstream of GFP is indicated by alternating red and gray boxes. The multiple cloning site of pPD Cloning a PCR amplified promoter: adapted restriction sites can be added to facilitate directional cloning. Generating a C-terminal translational fusion: the sequence of interest has to be cloned into the proper reading frame. The reading frame of pPD Vectors with shifted reading frames are also available pPD The reporter gene in pPD Other reporter genes are listed in Table 1.
The primary advantage of standard cloning approaches is the generation of a reusable reporter gene construct. Plasmids can be repeatedly amplified in bacteria and stored for long periods of time. This approach is referred to as PCR fusion.
For more details, see Figure 3 and Hobert The overlap is engineered into the fragments by the primers. By this method, a sequence of interest can be fused to any reporter gene.
Choose a reporter gene vector that suits the needs of the experiment. Remember that primer design depends upon the sequence of the reporter gene vector. This primer contains a linking sequence that overlaps with the reporter gene amplicon. The gene-specific primer linker should be at least 24 bp long and complementary to the MCS of the expression vector or to the coding region of the reporter gene. Inclusion of this intron greatly enhances reporter gene expression.
Figure 3. Generating a C-terminal translational PCR fusion. Primers A and B amplify the genomic region amplicon 1. Primer B adds a 24 bp overlap in frame to the GFP coding region. Primers C and D amplify the reporter gene e. The resulting fusion product amplicon 3 can be directly injected into C. Amplify reporter gene from expression vector with vector-specific primers for pPD The PCR reaction from Step 2 can be used directly for injection if the expected size fusion product is present in sufficient quantity.
If yield is low, the DNA sample can be concentrated by standard methods. PCR fusion technology is a good supplement to other strategies for the generation of reporter gene constructs.
The primary advantage to the PCR fusion approach is speed. The time required to make a PCR fusion product from start to finish is shorter than traditional cloning approaches. In addition, many reactions can be carried out in parallel. For the fluorescent protein-POI fusions, we generate straight fusions, without any linker.
When both orientations are available, a direct comparison of these fusion proteins may reveal which of the two better preserves the function of the POI. In the mVenus-APT1, a lipidation motif, which is needed for Golgi localization, is occluded and therefore this protein is mislocalized. The APT1-mVenus fusion is clearly a better choice to work with. We have encountered several proteins for which the N- and C-terminus of the POI are necessary for its native properties.
In these cases, an insertion of the fluorescent protein into the POI can work well since the N- and C-terminal residues of the fluorescent protein are relatively close together figure 1. Where should the fluorescent protein be inserted? Sites that are less likely to disrupt the structure of the POI would work best. Ideally, structural information can guide this decision. In our experience, the best site for the insertion is a loop in between secondary structure beta-sheets or alpha helices.
An additional requirement is that the site of insertion does not interact with other proteins or biomolecules. Although structural information may guide design, it is advisable to generate multiple constructs with different insertions, since it is difficult to predict which one will work as illustrated in figure 3 Mastop et al.
Unbiased, random insertions can be generated with a transposon-based strategy Sheridan, The introduction of DNA that encodes a fusion protein adds protein to the existing pool of protein within a cell and may lead to over-expression artifacts. This is an important downside of the technology and should always be considered when interpreting results.
However, it is advisable to first express the fusion protein from a plasmid-based system to assess whether the chimera is still functional. There are several choices to consider before generating a fusion protein, e. These choices will determine how well the fusion reflects the function of the native protein that is tagged.
The localization of the fusion protein can be verified and it can be compared to what is expected based on immunofluorescence or properties of the protein.
Determining other aspects of functionality catalytic activity or interactions can be challenging. Chalfie and his team found that Gfp gene produced GFP without added enzymes or substrates in both organisms.
The detection of GFP needed only ultraviolet light. Thereafter, many biologists introduced GFP into their experiments to study gene expression. Coli in Many scientists tried to mutate the Gfp gene to make the resultant protein react to wider wavelengths and emanate different colors. Other scientists studied different fluorescent proteins FPs. Roger Tsien, a professor at the University of California San Diego , in San Diego, California, reengineered the gene Gfp to produce the protein in different structures.
His team also reengineered other FPs. Due to Tsien's and other bioengineers' efforts, GFP could not only exhibit brighter fluorescence, but also respond to a wider range of wavelengths, as well as emit almost all colors, except for red. Tsien's findings enabled scientists to tag multiple colored GFPs to different proteins, cells, or organelles of interest, and scientists could study the interaction of those particles.
Other laboratories developed fluorescent sensors for calcium, protease and other biological molecules. Since then, scientists have reported more than distinct GFP-like proteins in many species. As GFP does not interfere with biological processes when used in vivo , biologists use it to study how organisms develop.
For example, after , Chalfie and his colleagues applied GFP in the study of the neuron development of C. In a paper, Chalfie and his colleagues describe how they first labeled a specific gene involved in tactile perception in neuron cells with GFP, and then observed the amount of fluorescence emitted by those cells. Because mutant cells produced less or more GFP than normal cells, the abnormal amount of fluorescence production indicated the abnormal development of mutants.
Since then, this field of research expanded to many other organisms, including fruitflies, mice, and zebra fish. Green Fluorescent Protein Green fluorescent protein GFP is a protein in the jellyfish Aequorea Victoria that exhibits green fluorescence when exposed to light. Ward, and Douglas C. Chalfie, Martin.
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