User:Jennyjsun/sandbox

The conceptual goal of this rotation project is to create a knock-in mouse line that can be used to visualize neuron morphology and distinct subcellular compartments in a genetically-encoded cell type.

Project Summary
The strategic route is outlined as follows:
 * 1) Research choices for the best florescent protein fusion.
 * 2) Determine the ideal target for visualization.
 * 3) Determine the best protein or polypeptide diffuse to the fluorescent protein to achieve subcellular targeting.
 * 4) Determine the fluorescent proteins with the best spectral separation for labeling cells.
 * 5) Determine if and what additional tags should be added for affinity purification.
 * 6) Research the best choice for building a polycistronic message
 * 7) IRES (internal ribosomal entry site) 2A protein skip, intein, others???
 * 8) Validate best choices for proteins and polycistronic messages in neuron culture.
 * 9) Do the fluorescent protein fusions target to the correct cell compartment, are they toxic?
 * 10) Does the polycistronic message strategy work. Do we get unwanted fusion proteins (example, we don't want our synapse marker to be linked, even at a low level, to our nuclear marker)?
 * 11) Build expression cassette and validate in culture.
 * 12) Is it cre and flp responsive? Does it express well?
 * 13) Build targeting vector and validate by sequence and restriction analysis. *TARGET POINT FOR THE END OF THE ROTATION
 * 14) Send off to core facility for ES work and screen colonies for correct integration site.
 * 15) Send off correct clones to core facility for blastocysts injection
 * 16) Breed out chimeras to produce a clean line.
 * 17) Activate line using TH_cre or DBH_cre and characterize the neuroanatomy and cellular compartmentalization of each fluorescent protein.

Targets
For our rotation goal, we want to choose three targets that structurally define neuron morphology; thus, we want to visualize the whole cell, the nucleus (for cell counts and possibly chromatin pulldowns), and synapses (to study synaptic contacts). Whole cell fluorescence is easily accomplished through the expression of a fluorescent protein. Nuclear visualization can be accomplished by fusing a fluorescent protein to a variety of nuclear proteins, including the commonly used H2B, one of five main histone proteins involved in the structure of chromatin. Synaptophysin, a synaptic vesicle protein, has been generally accepted as a presynaptic marker of synapses. Synaptophysin-GFP expression under a Cre driver line was recently used successfully in a mouse model and a synaptophysin-tdTomato mouse can be found at Jax Mice, with in situ staining indicating the presence of tdTomato in neurons. .

If this construct is successful, other potential targets to explore in the future include: the actin cytoskeleton (Lifeact), postsynaptic inputs (markers include PSD-95-excitatory glutamatergic; NMDA-R1; α-CAMKII; HOMER1-excitatory; and gephyrin-inhibitory GABAergic. Unfortunately, no universal postsynaptic markers labeling both inhibitory and excitatory are known), the plasma membrane, etc. Technologies such as L10 TRAP and Ribotag could serve to both label the whole cell and give insight into the transcriptome of specific cell types.

Fluorescent Proteins
In selecting fluorescent proteins, the following criteria were taken into consideration: intrinsic brightness (calculated as extinction coefficient * quantum yield * 1/1000; the brightness of EGFP), photostability, pH stability, maturation efficiency, monomer character, spectral overlap, and antibody existence. While the goal of this project is to create a mouse expressing three different colours (blue, green, and orange-red), fluorescent proteins were chosen through the visual and infrared spectrum to accommodate up to five and possibly more if UV-excitable and photoswitchable FPs are explored.

*Blue
mTagBFP is generally considered the most optimal blue fluorescent protein, mainly because it exists in monomer form, is the brightest (33) and most photostable in its class, and has a narrow emission spectrum (excitation peak at 399 nm, emission peak at 456 nm). . It has been used several times in multi-colour cell labeling to label the nucleus (H2B), in combination with TagGFP2, phiYFP, TagRFP, and mKate2 ; mCherry, mEGFP, and mTurquoise ; and mTurquoise, mEGFP, mKO2, and mKate2. Derived through mutagenesis from TagRFP, it is recognized by the anti-tRFP antibody. Recently, the group that developed TagBFP identified a single amino acid mutant (I174A) called TagBFP2, which has increased brightness (1.22x) and photostability.

Cyan
Until recently, mTurquoise was the optimal cyan fluorescent protein until a new EGFP cyan variant, mCerulean3 was created with increased brightness (35 vs 25) and more photostability Even more recently, mTurquoise was furthered mutated to create mTurquoise2, with "faster maturation, high photostability, longer mono-exponential lifetime and the highest quantum yield measured". However, because of their novelty, use of mCerulean3 has been sparse and use of mTurquoise2 nonexistent in published literature.

*Green
EGFP, Emerald, Sf-GFP, and TagGFP2 are the four most widely used green fluorescent proteins. Sf-GFP (super-folding GFP) has the highest brightness (54) and a high folding time but may have a tendency to dimerize and has not been widely used by researchers. Emerald also has a high brightness (39) and a higher folding time, but may bleach rapidly upon photostimulation. TagGFP2 has a similar brightness level to EGFP (34) but also possesses a faster maturation and higher pH stability. Since all are derivatives of GFP, they are recognized by anti-GFP antibodies and have excitation peaks around 485 nm and emission peaks around 509 nm. Sf-GFP can fold efficiently even when the fused protein fails to fold correctly, which may lead to increased background in the case of inaccurate targeting, but the 1.4x brightness advantage over Emerald makes it the most appealing candidate.

Yellow
mCitrine, mVenus, YPET (anti-YFP) mKO2 (anti-mKO2), mOrange (anti-mOrange)

*Orange
In the orange range, tdTomato, mKO2, and Tag-RFP-T are the most useful fluorescent proteins. tdTomato, at a brightness of 98, is the brightest FP of any colour, but exists as a tandem dimer than may interfere with localization of the fused protein. However, a Cre-driven synaptophysin-tdTomato fusion gene mouse line has been produced with successful synaptic expression. TdTomato can be detected with the dsRed antibody and has an emission peak of 554 nm and an excitation peak of 581 nm. Alternatively, both mKO2 and TagRFP-T are monomers, bright (37 and 34, respectively), photostable, and have fast maturation times.

Red
mCherry, mApple (anti-mCherry), mStrawberry (anti-mStrawberry)

Far-Red
mKate2 is across-the-board considered the best far-red fluorescent protein. mKate2 is a derivative of TagRFP and is recognized by the anti-tRFP antibody.

Others
T-Sapphire, mKeima

Conclusion
For a tricistronic cassette, TagBFP, Sf-GFP, and tdTomato are the best candidate fluorescent proteins, fulfilling the requirements of minimal spectral overlap, high brightness, and high photostability.





Additional Tags
Each of the three chosen fluorescent proteins have unique antibodies (TagBFP: anti-tRFP; Sf-GFP: anti-EGFP; tdTomato: anti-tdTomato). For additional binding specificity, antibodies against P2A have been used successfully in Western blot and immunoprecipitation. Because two P2A sites will be used, an additional tag will be added to one of the fusion proteins to distinguish between the P2A tags. A variety of affinity tags are both available and suitable, but we will go with c-myc because the tag is small (11 amino acids) and widely used in immunoprecipitation and Western blot technology.

Multicistronic Construction
There are two prominent strategies for construction of a multicistronic expression cassette: insertion of an internal ribosome entry site (IRES) between genes, and the linkage of genes through a virus-derived, "self-cleaving" 2A peptide. The main problems with IRES are its large size (>500 nts) and a significantly reduced expression of the second gene ; 2A peptides have recently emerged as a promising alternative. Several studies comparing IRES and 2A peptides have shown that use of 2A peptides results in higher downstream expression in differentiated cells, virus-injected mice and mice knock-in mouse lines. Occasionally the use of the 2A linker results in cleavage "skipping" and an unwanted fusion; however, adding a GSG linker between the N-terminus of the protein and the 2A peptide has been shown to improve cleavage significantly.

Several 2A peptides (T2A, P2A, F2A, and E2A) have been identified and there is no clear answer on which peptide is optimal for multicistronic expression in a knock-in mouse model. One study suggests that in a bicistronic vector expressed in cell culture, all four 2A peptides give equal expression levels of the post-2A EGFP expression reporter (as measured by fluorescence levels). Another suggests that P2A gives the highest expression in human cell lines, zebrafish embryos, and adult mice, as measured by western blotting and confocal microscopy. A third study demonstrated that a polycistronic cassette with 3 T2A sites linking four genes had the best expression in human embryonic stem cells, although they did not look at P2A. F2A was used successfully in a tricistronic vector in the knock-in mouse model, but the authors did not look at any of the other peptides. The authors also noted, importantly, that the use of EGFP as the post-2A expression reporter "substantially inhibited" the skipping ability of F2A, even with a GSG linker. Another group also used F2A tricistronically to express Cre and EGFP in a mouse line, and did not note any problems with the skipping ability.

Since we are unable to draw any conclusive directions from the literature, we have chosen P2A as our initial candidate for multicistronic cassette construction.

Insert Design
Our goal insert is as follows: H2B-TagBFP-cmyc-P2A-tdTomato-P2A-Synaptophysin-SfGFP.

GSG linkers included!

Insert Construction
H2B-TagBFP is available commercially, as well as tdTomato, a synapt, and sfGFP.

A synaptophysin-EGFP plasmid can be found here: http://www.addgene.org/26084/, with EGFP flanked by AgeI and EcoRV sites.

Construction of synaptophysin-sfGFP
Forward primer adding AgeI site: AGCGACCGGT AGCAAAGGAGAAGAACTTTTCAC (23 bp, Tm of 53.6°C) Reverse primer adding EcoRV site: CAGCGATATC TTAGTGATGGTGATGGTGATGG (22 bp, Tm of 54.5°C)

Isothermal cloning
Gibson, or isothermal cloning is a single step reaction that allows the assembly of multiple overlapping DNA fragments. To perform isothermal cloning, segments with overlapping regions of at least 40 base pairs are generated. For our construct, three segments will be used: Segments 1 and 2 overlap in the 33 bp of c-myc and first 7 bp of the GSG linker. Segments 2 and 3 overlap in bps 13-53 of the GSG/P2A region.


 * Segment 1: H2B-TagBFP + cmyc + first 7 bp of GSG linker
 * Segment 2: cmyc + GSG linker + P2A + tdTomato + first 57 bp of P2A
 * Segment 3: last 58 bp of P2A + synaptophysin-sfGFP

PCR amplification of Segment 1

 * Forward primer: ATGCCAGAGCCAGCGAAG (18 bp, Tm of 58.2°C)
 * Reverse primer adding the overlapping region: CGCTGCCCAGATCTTCTTCAGAAATAAGTTTTTGTTCCAT ATTAAGCTTGTGCCCCAGTTTG (22 bp, Tm of 58.6°C)

PCR amplification of segment 2
Because two P2A sites are being added to tdTomato, to prevent primer dimerization, PCR amplification must be done in two steps

Step 1 (adding the P2A + most of the cmyc site to the 5' end of tdTomato)
 * Forward primer: AAACTTATTTCTGAAGAAGATCTGGGCAGCGGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCT  ATGGTGAGCAAGGGCGAG (18 bp, Tm of 60.3°C)
 * Reverse primer: CTTGTACAGCTCGTCCATGCC (21 bp, Tm of 58.2°C)

Step 2 (adding the P2A to the 3' end of tdTomato and the rest of cmyc on the 5')
 * Forward primer: ATGGAACAA AAACTTATTTCTGAAGAAGATCTGGGC (27 bp, Tm of 55.4°C)
 * Reverse primer: AGGACCGGGGTTTTCTTCCACGTCTCCTGCTTGCTTTAACAGAGAGAAGTTCGTGGCGCCGCTGCC CTTGTACAGCTCCTGTACGC (20 bp, Tm of 56.0°C)

PCR amplification of segment 3

 * Forward primer: ACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCT  CTGCTGCTGGCAGACATGG (19 bp, Tm of 59.2°C)
 * Reverse primer: TTAGTGATGGTGATGGTGATGGGATC (26 bp, Tm of 58.3°C)

Change of plans: - synaptophysin-egfp plasmid found here: http://www.addgene.org/browse/sequence/12248/ - corresponds to mus musculus mRNA found here: http://www.ncbi.nlm.nih.gov/nuccore/1208520?report=fasta - paper from the creators can be found here: http://www.ncbi.nlm.nih.gov/pubmed/20634890

Assorted things were designed, primers added, etc.

Resources
Vista Browser Vista Enhancer Browser UCSC Mouse Genome Mouse Genome Informatics