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Here is the provenance of the components that we have used. For details of cloning, see the individual clone pages or the full methods paper. Here also is a list of relevant references.


[edit] Gateway components

The entry clones and destination vectors are all based on (three-insert) multisite Gateway technology from Invitrogen, specifically pDONR P4-P1R, pDONR221 (attP1-attP2), pDONR P2R-P3, and pDest R4-R3. Our understanding from Invitrogen is that they do not usually require any MTAs for clones generated by individual labs using their vectors. (You still need to buy recombination enzyme from them though!)

Sequence information all comes from the Invitrogen website. In some cases we have noticed point mutations in our constructs relative to their sequence. The most notable example is a point mutation in the ccdB coding sequence within the destination vector. In at least a couple of cases, we have seen a mutation in clones from different labs, leading us to believe that it derived from the original Invitrogen clone.

[edit] Fluorescent proteins (FPs)

[edit] EGFP

EGFP is the standard enhanced green fluorescent protein (1).

[edit] mCherry

mCherry is a monomeric red fluorescent protein, originally engineered by Tsien lab (2) from mRFP1, which in turn was a monomerized version of DsRed. The mCherry sequence we used was derived from Svoboda lab (3), and bears several silent mutations relative to the Tsien lab version.

[edit] Subcellular localization tags

[edit] nls-

The nuclear localization sequence is a 9 amino acid peptide (MAPKKKRKV) fused to the N-terminus of the fluorescent protein. The sequence was taken from the pCS2+nls vector.

[edit] -CAAX

The CAAX box (KLNPPDESGPGCMSCKCVLS.) encodes a 20 amino acid fusion plus stop codon to the C-terminus of the fluorescent protein. The sequence is from human H-ras (last 21 amino acids) and results in prenylation of the protein, targeting it to the plasma membrane (4).

[edit] H2A-

H2A encodes the zebrafish histone variant H2A.F/Z, which has previously been shown to label chromatin when fused to EGFP (5). H2A.F/Z was cloned from cDNA by Kristen Kwan, and fused to the N-terminus of mCherry.

[edit] IRES and polyA

[edit] encephalomyocarditis (EMCV) IRES

In mammalian systems, the most commonly used internal ribosome entry sequence (IRES) is that from encephalomyocarditis virus (EMCV). In zebrafish, the EMCV IRES has been shown to be active (6), but has not heretofore been widely used. The EMCV IRES sequence used here derives from the Clontech vector pIRES2-EGFP. The sequence is nearly identical to the “preferred IRES” sequence described by Bochkov and Palmenberg (7), beginning before the poly-C tract and extending to the 12th AUG, where we have placed the start codon of the EGFP or EGFP fusion. The EMCV IRES mRNA sequence contains 12 AUG codons, and is thought to form a complex secondary structure that somehow interacts with the ribosomal machinery to reinitiate translation at the 11th AUG (8).

Note that the IRES clones in Tol2kit v1.0 and 1.1 (clones 389, 390, and 391) contain seven As in the bifurcation loop; six As are likely to give more efficient translation of the second cistron (7). Tests of the A6 versions are under way (9/23/07).

[edit] SV40 late polyA signal

The SV40 late polyadenylation signal was derived from pCS2+. While many mammalian expression vectors use the SV40 early polyadenylation signal sequence, in Xenopus laevis and other systems the SV40 late polyA signal sequence has been shown to be much more effective in stabilizing mRNA transcripts and promoting translation (9,10).

[edit] Transposon backbone

The destination vectors in Tol2kit v1.2 (pDestTol2pA2/CG2) use the Tol2 backbone from pT2KXIGDin (11) from the Kawakami lab.

[edit] References

(1) Zhang G, Gurtu V, Kain SR. 1996. An enhanced green fluorescent protein allows sensitive detection of gene transfer in mammalian cells. Biochem Biophys Res Commun 227:707-711.

(2) Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY. 2004. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567-1572.

(3) Gray NW, Weimer RM, Bureau I, Svoboda K. 2006. Rapid redistribution of synaptic PSD-95 in the neocortex in vivo. PLoS Biol 4:e370.

(4) Moriyoshi K, Richards LJ, Akazawa C, O'Leary DD, Nakanishi S. 1996. Labeling neural cells using adenoviral gene transfer of membrane-targeted GFP. Neuron 16:255-260.

(5) Pauls S, Geldmacher-Voss B, Campos-Ortega JA. 2001. A zebrafish histone variant H2A.F/Z and a transgenic H2A.F/Z:GFP fusion protein for in vivo studies of embryonic development. Dev Genes Evol 211:603-610.

(6) Fahrenkrug SC, Clark KJ, Dahlquist MO, Hackett PB, Jr. 1999. Dicistronic Gene Expression in Developing Zebrafish. Mar Biotechnol (NY) 1:552-561.

(7) Bochkov YA, Palmenberg AC. 2006. Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location. Biotechniques 41:283-284, 286, 288 passim.

(8) Kaminski A, Howell MT, Jackson RJ. 1990. Initiation of encephalomyocarditis virus RNA translation: the authentic initiation site is not selected by a scanning mechanism. Embo J 9:3753-3759.

(9) Carswell S, Alwine JC. 1989. Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences. Mol Cell Biol 9:4248-4258.

(10) Matsumoto K, Wassarman KM, Wolffe AP. 1998. Nuclear history of a pre-mRNA determines the translational activity of cytoplasmic mRNA. Embo J 17:2107-2121.

(11) Urasaki A, Morvan G, Kawakami K. 2006. Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition. Genetics 174:639–649.

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