An Entry/Gateway® cloning system for general expression of genes with molecular tags in Drosophila melanogaster
© Akbari et al; licensee BioMed Central Ltd. 2009
Received: 05 August 2008
Accepted: 29 January 2009
Published: 29 January 2009
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.
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.
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.
Fusion proteins with molecular tags are widely used in biological studies. The most widely used are green fluorescent protein (GFP) and red fluorescent protein (RFP) for their visualization of tagged proteins in living cells. Other tags are also commonly used, such as the epitope tags myc, FLAG and HA. Each tag has its specific benefits and disadvantages, and it is desirable to generate multiple plasmids for producing various tagged versions of a protein. Occasionally, a tagged protein might function differently than the original protein. To resolve this potential problem, one may place the tag at either the N-terminal or C-terminal region of the protein or try other tags in a trial-and-error manner. A series of destination vectors containing various combinations of a promoter and molecular tags can greatly reduce the time and labor of creating the required plasmids in these applications. In Drosophila, the P-element based UAS/Gal4 system is widely used for expression of transgenes due to its extremely versatile nature . An extensive set of UAS P-element destination vectors have been created previously, for example, the pPWG and pPWR vectors, which can express the eGFP- or mRFP-tagged fusion proteins (T. Murphy personal communication, http://www.ciwemb.edu/labs/murphy/Gateway%20vectors.html and https://dgrc.cgb.indiana.edu/vectors/store/vectors.html?product_category=6). The UAS/Gal4 system, however, may cause over-expression of the desired protein, which often induces unexpected effects when dosage-sensitive genes are studied, for example the chromatin insulator protein CP190 described in this study. We have developed, based on the Entry/Gateway® strategy, a cloning system that uses the ubiquitin (Ubi-63E) promoter to express genes at lower and tolerable levels and can be used to quickly generate plasmids for expressing tagged fusion proteins in Drosophila melanogaster.
Chromatin insulators are a class of regulatory DNA elements in the genome. Protein complexes assembled on a chromatin insulator sequence can insulate enhancers from a promoter when the insulator is located between the enhancers and the promoter. Proposed functions of chromatin insulators include organizing boundaries of chromatin sub-domains and regulating local gene expression by interfering with enhancer-promoter interactions. It has been hypothesized that chromatin insulator complexes, bound at distal genetic locations, may "aggregate" to form structures named "insulator bodies"[2, 3]. We have used the developed cloning system to express CP190 in Drosophila melanogaster. CP190 is a nuclear protein in the fly and is a shared essential component of several kinds of chromatin insulator complexes [3–5]. The fluorescence of the CP190-eGFP or CP190-mRFP proteins allows us to monitor the distribution of related chromatin insulators in living cells. We discovered that the distribution of CP190-related chromatin insulators varied significantly in living cells from separate tissues. We also observed significant movement of CP190-containing insulator bodies in living cells, which resulted in the fusion of smaller aggregates into larger complexes. This activity likely reflects the alterations of the organization of chromatin higher-order structure in these cells.
A vector for generating entry clones using TA-based methods
A set of P-element based Gateway® destination vectors for general expression of epitope-tagged fusion protein in flies
The features of the P-DEST vectors and pGWS.
P-element destination vector
P-element destination vector
P-element destination vector
P-element destination vector
P-element destination vector
P-element destination vector
TA Entry Vector
Over-expression of CP190 using the Gal4/UAS system causes lethality or developmental defects
Expression of CP190 using P-DEST vectors results in normal flies
Formation of "insulator bodies" in CP190mRFP-expressing cells
The genome projects of Drosophila melanogaster and of other species have revealed many genes that were not investigated before. Antibodies for the encoded proteins of these genes, in many cases, are not yet available. In such cases, it can be difficult to determine the biological activities of these proteins inside cells. Convenient methods for rapidly generating epitope-tagged transgenic Drosophila melanogaster lines will facilitate comprehensive in vivo functional analysis of these uncharacterized genes. CP190 is a housekeeping nuclear protein with general functions in chromatin organization. It is expressed in cells of all tissues in Drosophila melanogaster. Expression of CP190 in flies using the currently available Gal4/UAS induction system resulted in lethality or missing tissues. These unexpected results indicate that the development of Drosophila melanogaster is sensitive to the expression levels of CP190. We hypothesized that the available Gal4 drivers might have induced intolerably high expression levels of CP190 in cells from the UAS-CP190 transgene, thus leading to the developmental defects. We successfully corrected this over-expression problem by using the Ubi-63E promoter encoded in the P-DEST vectors. The Ubi-63E promoter has been characterized well previously [6, 10–12]. The promoter can drive ubiquitous expression of a transgene in cells of most tissues. In addition, its temperature-sensitive feature allows for adjustment of the expression levels by heat treatment. Flies expressing CP190 driven by Ubi-63E promoter are healthy and the expression rescued the lethality of the CP190 homozygous mutant. These results suggest that the promoters of CP190 and Ubiquitin may have similar strengths and may be interchangeable. Our results indicate that the P-DEST vectors allow the expression of CP190, a dosage-sensitive housekeeping gene, at tolerable levels in many tissues. These vectors will be valuable for studies of other proteins with similar dosage sensitivity. The six P-DEST vectors described in this paper provide time-saving tools for ubiquitous expression of fusion proteins, with an N-terminal or C-terminal epitope-tag, in Drosophila melanogaster. Recently, new technologies in transgenic vector design have been developed, such as the "MultiSite Gateway system"  and the attB site of the phiC31 system. Incorporating these technologies into the P-DEST vectors in the future will make this vector system more flexible and easier to use.
Cloning of unstable DNA sequences, such as DNA with multiple repeated sequences or inverted repeats, is often performed in the SURE ® strain of E. coli (Stratagene) which is Kan resistant. Currently, a few vectors are commercially available for generating entry clones. They all use Kan as the selectable marker, such as pENTR™ vectors (Invitrogen). These vectors, thus, cannot be used for cloning with the SURE ® strain of E. coli. The newly-designed pGWS uses Gen as the selectable marker, thus avoiding this problem. The pGWS is unique from its parental plasmid pGWG, which uses an AhdI digestion to generate the 3' "T" overhangs for TA cloning. The AhdI-digested pGWG often loses the 3' "T" overhangs due to undefined exonuclease activities during the AhdI digesting reaction, causing low cloning efficiencies (data not shown). The redesigned pGWS significantly improves the cloning efficiency compared to the original pGWG vector.
Using P-DEST vectors, we have generated transgenic flies expressing CP190 proteins tagged with eGFP or mRFP. The CP190mRFP or CP190eGFP proteins can rescue the defective insulator function in homozygous CP190 mutations, suggesting that the mRFP- and eGFP-tagged CP190 proteins are fully functional in flies. The fluorescence of these tagged CP190 proteins allows us to view dynamic changes in the distribution of chromatin insulators in living cells. We detected, similarly to the antibody staining results published previously , the formation of insulator bodies in living cells. The number and sizes of insulator bodies, however, vary among cell types. Generally, insulator bodies are larger in cells that have fewer insulator bodies. This phenomenon suggests that large insulator bodies may be assembled from smaller ones. By monitoring the movement of CP190mRFP in living cells, we observed events of fusion, or assembly, of CP190-containing particles. These events reflected the reorganization of insulator bodies, and likely chromatin reorganization too, in the nucleus during the time of examination. The assembly and disassembly of insulator bodies appear to be normal activities within the cell nucleus, and, in addition, may be regulated differently in many cell types. Since most of the CP190mRFP fluorescent signals in polytene cells are associated with polytene chromosomes as many bands, it is likely that the majority of the CP190mRFP proteins in the diploid cells are also associated with DNA-bound chromatin insulator complexes. If most CP190 proteins are in DNA-bound complexes, the formation of big insulator bodies would be creating a higher-order chromatin structure that allows for the association of multiple insulator complexes at distant genetic locations. On the other hand, it is possible that the big insulator bodies are insulator complexes dissociating from a number of sub-regions of chromatin due to unknown regulatory mechanisms. It will be interesting to determine how cells with one pattern of chromatin insulator body distribution may be induced to change into another pattern and whether the rearrangement reflects, or causes, changes in local transcriptional activities. The assembly and disassembly of insulator bodies may be regulated via, for example, modifying the proteins in the insulator complexes to establish alternative higher-order structures of chromatin in separate cell types.
We have developed a convenient cloning system using the Entry/Gateway® technology. The cloning system includes one vector for generating entry clones and six P-element destination vectors (P-DESTs) for expressing fusion proteins in Drosophila melanogaster. The pGWS vector provides a non-commercial alternative method for creating entry clones. The vector will be particularly useful for cloning unstable DNA sequences using the SURE ® strain of E. coli. The six P-DEST vectors contain the Ubi-63E promoter, which can drive the expression of transgenes in many tissues in transgenic flies at physiological, or at least tolerable, levels. Each P-DEST also encodes one of the molecular tags that may be fused to either the N- or C-terminus of the transgenic protein. The pGWS and six P-DEST vectors provide time-saving tools for ubiquitous expression of fusion proteins in Drosophila melanogaster.
We have used the P-DEST vector system to express in flies the mRFP- or eGFP-tagged CP190, which is a shared essential component of multiple kinds of chromatin insulator complexes and is one of the dosage-sensitive housekeeping genes. The expressed CP190 fusion proteins function similarly to the wildtype CP190 protein. The fusion proteins associate with polytene chromosomes as multiple bands in living polytene cell nuclei. On squashed polytene chromosome samples, the tagged CP190 protein co-localizes with other proteins of the gypsy insulator complex at gypsy inserted loci. In living diploid cell nuclei, the fusion protein localizes to particles of various sizes, termed previously as "insulator bodies". By monitoring the fluorescent signals of CP190mRFP in living cells, we have found that CP190-containing insulator complexes are moving in the nucleus. In addition, we observed events of fusion, which presumably correlates to assembly, of CP190-containing insulator bodies of various sizes. This movement of insulator complexes may be a result of the altered organization of chromatin higher order structure. Our results indicate that the assembly and disassembly of insulator bodies are normal and dynamic activities in living cells.
All fly stocks were maintained in 23°C or 26°C environmental insect culture chambers. The P-elements encoding tagged-CP190 were introduced into flies by the traditional germ-line transformation method. The pPWG and pPWR vectors were obtained from Drosophila Genomic Resource Center. The act5c > Gal4, ey > Gal4, dpp blk > Gal4, and hs70 > Gal4 flies were obtained from Bloomington stock center.
Tissue preparation, staining, microscopy and image processing
The eGFP- or mRFP-CP190 expressing larvae were viewed under a Leica MZ16 stereoscope and imaged using a Leica FX300 digital camera. For live insulator body imaging, eGFP- or mRFP-CP190-expressing tissues were dissected in phosphate saline. The dissected tissues were viewed immediately after dissection under a Leica DM5500 microscope and were imaged using a Leica FX350 digital camera. The spread polytene chromosomes were prepared and stained with indicated antibodies using a method described previously . Rabbit-anti-GFP antibody (Invitrogen A11122) was used at 1:500 dilution. Mouse-anti-RFP antibody (Abcam) was used at 1:400 dilution. Adult flies were viewed under Leica S8 stereoscope. Images were taken by Leica DFC280 digital camera. Multiple pictures of one individual fly or of a tissue may be taken and overlaid for obtaining better depth of field. The image-overlay was processed automatically by Helicon Focus (Helicon Soft Ltd).
The P-destination vectors were created using CaSPeR4 as the backbone. The KpnI-PstI fragment containing the Ubi-63E promoter in pWUM6 was inserted into the Kpn1/Pst1 sites of CaSPeR4 to become pP [CaSU]. The SV40(A) fragment was PCR amplified from vector pAWG (T. Murphy, unpublished results, obtained from the Drosophila Genomics Research Center) using the primer pair (forward primer 5'-GCGGCCGCCTAGCAGGATCTTTGTGAAG-3', reverse primer 5'-GCGGCCGCTGTTGAATACTCATACTCTTCC-3'). The resulting 976 bp fragment was digested with NotI and was inserted into the NotI site of pP [CaSU] to make p [CaSU(A)]. The gateway cassettes were digested from vectors pAGW, pAWG, pAMW, pAWM, pARW, pAWR (T. Murphy, unpublished results; obtained from the Drosophila Genomics Research Center) using restriction sites EcoRV/NheI and cloned into StuI/XbaI sites of p [CaSU(A)] to become pUGW, pUWG, pUMW, pUWM, pURW, pUWR respectively. For creating pGWS, pGWG was PCR amplified with the primer pair GWS_F (5'-GGGGTAAGTCTCTAGACCCAGCTTTC-3') and GWS_R (5'-GGGAAGTCAAAGCCTGCTTTT-3'). The resulting fragment was self-ligated with T4 ligase (NEB) and was propagated in the SURE strain of E. coli. To generate a TA-cloning vector from pGWS, pGWS DNA was digested with SmaI. The linearized DNA was recovered and a "T" was added at the 3' end of each strand by incubating the DNA with 2 mM of dTTP and Taq polymerase at 72°C for 2 hours. For generating the eGFP entry clone from pGWS, the eGFP fragment was PCR amplified from pUWG using the primer pair GFP_F (5'-CTTGTACAGCTCGTCCATGC-3') and GFP_R2 (5'-CTTGTACAGCTCGTCCATGC-3'). The resulting fragment was mixed with the pGWS TA vector DNA, prepared as described above, and was ligated with T4 ligase at 12°C overnight. The ligated product (pGWS.GFP) was propagated in the Mach1 strain (Invitrogen) of E. coli. For generating the His-tagged GFP fusion protein, the Clonase II® reaction of pGWS.GFP and pDEST17 (Invitrogen) was performed following the instructions from Invitrogen.
enhanced green fluorescent protein
monomeric red fluorescent protein
We would like to thank Jeff Sekelsky for providing us with the pWUM6 vector. Also, we would like to thank Dr. T. Murphy for providing the pAWG, pAGW, pAMW, pAWM, pARW, pAWR, pPWG, pPGW, pPMW, pPWM, pPWR, and pPRW plasmids. We thank Grant Mastick, Thomas Kidd, Patricia Berninsone, and Terence Murphy for reading the manuscript and providing valuable opinions. This work was supported by the NSF grant MCB0639945 and was made possible by NIH Grant Number P20 RR-016464 from the INBRE Program of the National Center for Research Resources.
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