An Entry/Gateway cloning system for general expression of genes with molecular tags in Drosophila melanogaster

Abstract

Background: Tagged fusion proteins are priceless tools for monitoring the activities of biomolecules in living cells. However, over-expression of fusion proteins sometimes leads to the unwanted lethality or developmental defects. Therefore, vectors that can express tagged proteins at physiological levels are desirable tools for studying dosage-sensitive proteins. We developed a set of Entry/Gateway vectors for expressing fluorescent fusion proteins in Drosophila melanogaster. The vectors were used to generate fluorescent CP190 which is a component of the gypsy chromatin insulator. We used the fluorescent CP190 to study the dynamic movement of related chromatin insulators in living cells.

Results: The Entry/Gateway system is a timesaving technique for quickly generating expression constructs of tagged fusion proteins. We described in this study an Entry/Gateway based system, which includes six P-element destination vectors (P-DEST) for expressing tagged proteins (eGFP, mRFP, or myc) in Drosophila melanogaster and a TA-based cloning vector for generating entry clones from unstable DNA sequences. We used the P-DEST vectors to express fluorecent CP190 at tolerable levels. Expression of CP190 using the UAS/Gal4 system, instead, led to either lethality or underdeveloped tissues. The expressed eGFP- or mRFP-tagged CP190 proteins are fully functional and rescued the lethality of the homozygous CP190 mutation. We visualized a wide range of CP190 distribution patterns in living cell nuclei, from thousands of tiny particles to less than ten giant ones, which likely reflects diverse organization of higher-order chromatin structures. We also visualized the fusion of multiple smaller insulator bodies into larger aggregates in living cells, which is likely reflective of the dynamic activities of reorganization of chromatin in living nuclei.

Conclusion: We have developed an efficient cloning system for expressing dosage-sensitive proteins in Drosophila melanogaster. This system successfully expresses functional fluorescent CP190 fusion proteins. The fluorescent CP190 proteins exist in insulator bodies of various numbers and sizes among cells from multiple living tissues. Furthermore, live imaging of the movements of these fluorescent-tagged proteins suggests that the assembly and disassembly of insulator bodies are normal activities in living cells and may be directed for regulating transcription.

Figure 1

The Entry/Gateway^® cloning procedures for generating epitope-tagged fusion proteins using the P-element destination vectors. The procedures of Entry/Gateway^® cloning are illustrated in the diagram as two major steps. (1) In the first step, a fragment encoding the open reading frame (ORF) is inserted into an entry vector to generate entry clones. Two entry vectors were described in this study: (i) the pGWS which uses a TA-based method; (ii) pENTR/D-TOPO (Invitrogen) which uses a TOPO-based method. (2) In the second step, the ORF in the entry clone is recombined into one of the P-DEST vectors.

Figure 2

Structure of the pGWS and P-destination vectors. (A-B) The detailed structure of P-DEST vectors with N-terminal tags (A), and with C-terminal tags (B). The sequences and the reading frame from the epitope tags are shown below the map. The attR1 and attR2 sequences for Clonase II^® recombination are shaded. (C) The structure and sequences of the pGWS. The circular pGWS has a unique SmaI site which is a restriction enzyme that can cut pGWS into a linear DNA with two blunted ends. The 3'-protruding ends after Taq T-tailing for TA cloning are shown in brackets. The reading frame after LR recombination with P-DEST vectors is indicated.

Figure 3

The pGWS entry clones are efficient in the Clonase II LR reactions. (A) The diagram of LR reaction of the pGWS.eGFP entry clone and pDEST17. Shown are features of the two parental plasmids and the recombined product encoding the eGFP protein with poly-His tag. The arrows indicate the primers for determining the bacteria colonies that contain the correctly recombined plasmid. (B) Colonies of the eGFP-His expressing bacteria are fluorescent. (C) The bacterial lysate and the His-bind-chromatography-purified eGFP-His protein were analyzed by SDS-PAGE and stained with Coomassie blue. The arrow points to the purified His-eGFP protein.

Figure 4

Morphological phenotypes of CP190 over-expressingflies. Morphological phenotypes of the Ey > Gal4/UAS-CP190mRFP fly (A and D), the dpp^blk > CP190mRFP fly (B and E), and the UAS-CP190mRFP fly (C and F). (A and D) In Ey > Gal4/UAS-CP190mRFP flies, the eyes were not developed (A, arrowhead). The legs are normal (D, arrow). (B and E) In the dpp^blk > Gal4/UAS-CP190mRFP flies, the eyes were developed, but were slightly rough (B, arrowhead). The distal parts of the legs, including tarsal and claw segments were underdeveloped or missing (B and E, arrows). (C and F) In the UAS-CP190mRFP flies, the eyes are normal (C, arrowhead). The legs are normal (F). All arrows point to the first tarsal segment of the first leg (B, D, E, and F).

Figure 5

Expression of GFP- or mRFP-tagged CP190 in transgenic flies. (A-B) A third instar larva expressing eGFP-tagged CP190 (A), or mRFP-tagged CP190 (B). (C-D) Fluorescent signals of CP190eGFP in a salivary gland (C), and in the nucleus of a salivary gland cell (D). (E-F) Fluorescent signals of CP190mRFP in a salivary gland (E), and in the nucleus of a salivary gland cell (F). (G-I) The distribution of CP190mRFP (G) and Mod67.2 (H) proteins at the tip of X chromosome. The polytene chromosome was prepared from a y² 3^rd instar larva. The white arrows point to the y locus where contains a copy of the gypsy insulator. The red arrow point to the location that contain only CP190 protein but do not contain Mod67.2 protein.

Figure 6

Dynamic distribution of CP190-containing chromatin insulators in living cells. (A-F) Fluorescent signals of CP190mRFP in living imaginal disc cells. (G-I) Movement of chromatin insulator bodies shown by CP190mRFP time-lapse images taken at the indicated times. Arrows point to insulator bodies which are moving toward each other (G and H) and the fused large insulator bodies (I).