construction of heart vectors

pBleeding Heart construction
The vectors are based on the tol2 vector T2KSAG (ref 1). We designed the vectors to maintain unique sites at as many junctions as possible to allow for simple swapping of componenets. Vector construction was done as follows (flow diagram):

1) We first deleted ~2 kb of the Medaka tyrosinase gene which is present in the T2KSAG back bone to create T2DT.

2) Several small cardiac promoters were placed in front of the fluorescent protein cherry. We found that expression from the cardiac myosin light chain (cmlc2; ref 2) could easily be detected at 2.5 dpf when injected into one cell embryos at 25 ng/ul. To add the visible reporter to the modified tol2 vector, we took advantage of the fact that the SV40 poly adenylation signal is bidirection and inserted a cmlc2 cassette driving the fluorescent protein cherry (ref 3) distal and in the opposite orientation of the GFP in T2DT to create pBleeding Heart 2 (pBH2 for short).

3) We replaced the splice acceptor/GFP cassette ( derived from KSAG; ref 1) with a custom designed polylinker to create pBH mcs.

4) We added YFP from eYFP-N1 (ref 4) with and without a C-terminal custom designed polylinker to create pBH mcs YFP and pBH YFP mcs.

5) We added a GAL4 UAS (gal4 binding site x 11; ref 5) to the YFP vectors to create pBH UAS mcs YFP and pBH UAS YFP mcs.

6) We inserted a Gateway cassette (ref 6) into each of these vectors to create pBH Gtwy mcs YFP, pBH mcs YFP-Gtwy, pBH UAS Gtwy YFP, and pBH UAS YFP Gtwy.

pCold Heart construction

7) We swapped out cherry from pBH mcs for cerulean (ref 7) to create pCH mcs.

8) We added GAL4VP16 (ref 8) and miniGAL4-9 (a.a. 1-168,727-881; ref 9) cassettes to created pCH mcs G4VP16 and pCH mcs G4m.

9) We inserted a gateway cassette in both vectors to create pCH Gtwy G4VP16 and pCH Gtwy G4m.

pGep Heart construction

10) We swapped out cherry from pBH mcs for eYFP to create pGH mcs.

References

1) Kawakami K, Takeda H, Kawakami N, Kobayashi M, Matsuda N, Mishina M. A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev Cell. 2004 Jul;7(1):133-44.

2) Huang CJ, Tu CT, Hsiao CD, Hsieh FJ, Tsai HJ.Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish. Dev Dyn. 2003 Sep;228(1):30-40.

3)Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. NAt Biotechnol. 2004 Dec;22(12):1567-72.

4) from Clontech, Inc.

5) The sequenced UAS contains 11 copies of the GAL4 binding domain. The UAS was derived from pCS2-GAL4VP16 obtained from R. Wong Lab (WUSTL) which originally contained 14 sites. This UAS repeat appears to be somewhat unstable.

6) Walhout AJ, Temple GF, Brasch MA, Hartley JL, Lorson MA, van den Heuvel S, Vidal M. GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol. 2000;328:575-92.

7) Rizzo MA, Springer GH, Granada B, Piston DW. An improved cyan fluorescent protein variant useful for FRET. Nat Biotechnol. 2004 Apr;22(4):445-9.

8) Sadowski I, Ma J, Triezenberg S, Ptashne M. GAL4-VP16 is an unusually potent transcriptional activator. Nature. 1988 Oct 6;335(6190):563-4.

9) Ding WV, Johnston SA. The DNA binding and activation domains of Gal4p are sufficient for conveying its regulatory signals.Mol Cell Biol. 1997 May;17(5):2538-49.