Construction and characterization of new piggyBac vectors for constitutive or inducible expression of heterologous gene pairs and the identification of a previously unrecognized activator sequence in piggyBac
© Shi et al; licensee BioMed Central Ltd. 2007
Received: 18 September 2006
Accepted: 18 January 2007
Published: 18 January 2007
We constructed and characterized several new piggyBac vectors to provide transposition of constitutively- or inducibly-expressible heterologous gene pairs. The dual constitutive control element consists of back-to-back copies of a baculovirus immediate early (ie1) promoter separated by a baculovirus enhancer (hr5). The dual inducible control element consists of back-to-back copies of a minimal cytomegalovirus (CMV min ) promoter separated by a synthetic operator (TetO7), which drives transcription in the presence of a mutant transcriptional repressor plus tetracycline.
Characterization of these vectors revealed an unexpected position effect, in which heterologous genes adjacent to the 3'- terminal region ("rightward" genes) were consistently expressed at higher levels than those adjacent to the 5'-terminal region ("leftward" genes) of the piggyBac element. This position effect was observed with all six heterologous genes examined and with both transcriptional control elements. Further analysis demonstrated that this position effect resulted from stimulation of rightward gene expression by the internal domain sequence of the 3'-terminal region of piggyBac. Inserting a copy of this sequence into the 5'- terminal repeat region of our new piggyBac vectors in either orientation stimulated leftward gene expression. Representative piggyBac vectors designed for constitutive or inducible expression of heterologous gene pairs were shown to be functional as insect transformation vectors.
This study is significant because (a) it demonstrates the utility of a strategy for the construction of piggyBac vectors that can provide constitutive or inducible heterologous gene pair expression and (b) it reveals the presence of a previously unrecognized transcriptional activator in piggyBac, which is an important and increasingly utilized transposable element.
piggyBac is a class II transposable element that was originally discovered as the IFP2 element from the lepidopteran insect cell line, TN-368, due to its tendency to insertionally inactivate certain baculovirus genes to produce mutants with a distinctive plaque phenotype known as Few Polyhedra [1, 2]. Functionally, piggyBac encodes a transposase with a precise cut and paste mechanism and a unique preference for TTAA sites [3–6]. Thus, it is considered to be the type element of the TTAA-specific transposon family . Structurally, piggyBac is a 2.4 kb DNA molecule with a single 1.8 kb open reading frame that terminates on both ends with 13 bp perfect inverted terminal repeat domains (TRD's). piggyBac also has two additional 19 bp subterminal inverted repeats located asymmetrically 31 bp from the 5'-TRD and 3 bp from the 3'-TRD . Sequences similar to the piggyBac open reading frame have been identified in all animal species for which extensive genomic sequences are available, including the human. However, most appear to be either incomplete or interrupted and, therefore, probably do not encode functional transposons .
The mobility and transposition functions of piggyBac have been established and exploited to develop an important binary system for insect germline transformation . This system consists of a DNA vector, which can be mobilized due to the presence of the piggyBac 5'- and 3'-TRD sequences, and a helper plasmid, which encodes the transposase. The vector also includes a promoter, which controls transcription of an inserted, heterologous gene of interest, and a whole-body or eye color marker, which can be used to identify transgenic offspring.
The Mediterranean fruit fly was the first target organism to be successfully transformed using the piggyBac system  and it has subsequently been used to transform a wide variety of insects (reviewed in reference ). Recently, the piggyBac system has been used to transform many other types of organisms ranging from the protist, Plasmodium falciparum  to the mouse, Mus musculus . Thus, piggyBac is widely and increasingly recognized as an important tool for genetic transformation in many different biological systems.
Current piggyBac vectors are designed to introduce a single heterologous gene of interest into the genome of a target organism, in addition to the marker gene. However, some transgenic approaches require the introduction of multiple heterologous genes of interest into a single target organism. To meet this requirement, we constructed a new set of piggyBac vectors designed to simultaneously transfer pairs of heterologous genes placed under the control of dual constitutive or regulated transcriptional elements, which included duplicate promoters in a back-to-back configuration. One advantage of this approach is that it allowed us to couple both promoters to a single enhancer or regulatory domain, which minimized the overall size of the transcriptional control region.
During the process of characterizing the induction of heterologous gene expression in lepidopteran insect cells by these new piggyBac vectors, we discovered an unexpected position effect, in which the rightward-oriented heterologous genes were consistently expressed at higher levels than the leftward-oriented ones. Further analysis revealed that this was the result of stimulation by a previously unrecognized activator element in the 3'-TRD of piggyBac. We subsequently duplicated this activating sequence and used it to balance expression of the rightward and leftward heterologous genes in our new vectors. Thus, this study yielded not only a substantial set of new piggyBac vectors, but also provided new basic information on this important transposable element, both of which will be of general interest to the biomedical research community.
Results and Discussion
New piggyBac vectors for transfer and constitutive or inducible expression of heterologous gene pairs
An unexpected position effect on heterologous gene expression by the new piggyBac vectors
The molecular cloning schemes used in this study initially yielded four sets of piggyBac vectors in which six different heterologous genes were placed under the control of the constitutive or inducible regulatory elements described above (Fig. 4). Six of these vectors had the three "Glyco-A" genes (GnTII, ST6GalI, and SAS) with a rightward orientation (Fig. 4, Sets 1 and 3), while the other six had these same genes with a leftward oriention (Fig. 4, Sets 2 and 4) with respect to the 5'- and 3'-TRD elements of piggyBac, as defined by Li and coworkers .
The position effect does not reflect antisense down-regulation of the leftward genes
Together, the results of the transient expression assays performed using the piggyBac vectors with BGHpolyA insertions or transposase promoter deletions strongly suggested that antisense transcription originating in the piggyBac transposase promoter within the 5'-TRD is not responsible for the observed position effect.
The position effect reflects activation of the rightward genes
A previously unrecognized activator sequence in the piggyBac 3'-TRD
Functionality of dual piggyBac vectors in transgenic insects
The initial purpose of this study was to produce and characterize new piggyBac vectors that could be used to transfer heterologous gene pairs into the genome of a target organism in either constitutively- or inducibly-expressible forms. Functional characterization of these new vectors revealed an unexpected position effect that was independent of the identity of the heterologous gene or the transcriptional control element. In the process of performing experiments designed to help us understand the underlying reason for this position effect, we discovered a previously unrecognized cis-activating element derived from the internal domain of the 3' terminal repeat in the piggyBac transposable element. This element appears to function as an enhancer element, as it stimulates heterologous gene expression in an essentially orientation-independent fashion, albeit to different levels. However, formal definition of this element as an enhancer would require additional characterization to determine if it can function autonomously in a position- and orientation-independent fashion.
The discovery of this element will be of general interest to investigators who are aware of piggyBac and its widespread and growing applications as a transformation vector. In addition, given their ability to provide constitutive or inducible expression of heterologous gene pairs, the large set of new dual piggyBac vectors described in this study will be of interest to investigators who need to introduce multiple genes into a single target organism.
High fidelity KOD (Novagen, Madison, WI) DNA polymerase was used as described in the manufacturer's manual. Briefly, 50 μL PCR reactions consisted of 5 μL of 10X KOD DNA polymerase buffer, 5 μL of dNTP mix (10 mM each), 2 μL of 25 mM MgCl2, 0.5 μL of KOD DNA polymerase, 0.5 μL of each primer (50 μM), 10 μL of template, and 26.5 μL of H2O. The fragments were amplified after an initial denaturation step at 95°C for 2 min using 30 cycles of 15 sec at 98°C, 30 sec at appropriate primer annealing temperatures, and 1.5 min at 72°C. The desired fragments were purified by agarose gel fractionation before being cloned into either pCRBluntII-TOPO (Invitrogen, Carlsbad, CA) or pCR2.1-TOPO (Invitrogen), and sequence-verified clones were used to assemble all of the final constructs described in this study.
General molecular cloning methods were performed as described in reference . PCR products were cloned into pCRBluntII-TOPO (Invitrogen) or pCR2.1-TOPO as described by the manufacturer. All restriction endonucleases were purchased from New England Biolabs, Inc. (Beverly, MA).
Construction of piggyBac vectors for constitutive expression of heterologous gene pairs
Oligonucleotides used in this study
Sequence (5' to 3')
In a parallel set of cloning reactions (Fig. 1B), a DNA fragment containing the bovine growth hormone polyadenylation signal  was PCR amplified using pCR3.1 (Invitrogen) as the template and BGHsense and BGHanti as the primers (Table 1). The product was cloned into pCRBluntII-TOPO and an error-free clone identified by restriction mapping and DNA sequencing was designated pBGHpolyA-TOPO.1. The BGHpolyA fragment was then excised from pBGHpolyA-TOPO.1 with ApaI and BsaI and subcloned into the same sites of pDIE1-TOPO.2 to produce pDIE1-TOPO.3.
A third parallel set of cloning reactions (Fig. 2A) was performed to place three different fluorescent marker genes under the transcriptional control of the eye-specific promoter, 3xP3 , for subsequent insertion into pDIE1-TOPO.3. First, the 3xP3 promoter fragment was PCR amplified using pBSII-LTR1.1k-ECFP  as the template and 3xP3sense plus 3xP3anti as the primers (Table 1). The product was cloned into pCRBluntII-TOPO, an error-free clone designated p3xP3-TOPO.1 was identified by restriction mapping and DNA sequencing, and then the ApaI and BsaI fragment of pBGHpolyA-TOPO.1, which contains the BGH polyA signal, was inserted at the corresponding sites of this plasmid to produce p3xP3-TOPO.2. Subsequently, each fluorescent marker gene of interest, including DsRed1, EGFP, and EYFP, was individually PCR-amplified using p3xP3-DsRed1-ORF (Li and Fraser, unpublished), pBSII-LTR1.1k-ECFP , and pBSII-LTR1.1k-EYFP  as the templates and DsRed1sense plus DsRed1anti, EGFPsense plus EGFPanti, and EYFPsense plus EYFPanti (Table 1) as the primers, respectively. Each individual PCR product was cloned into pCRBluntII-TOPO to produce pDsRed1-TOPO.1, pECFP-TOPO.1, and pEYFP-TOPO.1, respectively, and error-free clones were identified by restriction mapping and DNA sequencing. Finally, each individual marker was excised with NheI and ApaI and subcloned into the corresponding sites of p3xP3-TOPO.2 to produce p3xP3DsRed1-TOPO.2, p3xP3ECFP-TOPO.2, and p3xP3EYFP-TOPO.2.
The eye color markers were subsequently incorporated into the intermediate plasmids containing the dual constitutive ie1-hr5-ie1 transcriptional control element (Fig. 2B). The red, cyan, and yellow fluorescent protein markers were excised by digesting 3xP3DsRed1, 3xP3ECFP, and 3xP3EYFY with BglII and BsaI and each was individually subcloned into the BamHI and BsaI sites of pDIE1-TOPO.3 to produce pDIE1DsRed1-TOPO.3, pDIE1ECFP-TOPO.3, and pDIE1EYFP-TOPO.3, respectively. Subsequently, the KpnI-HindIII fragment from pBGHpolyA-TOPO.1 was subcloned into the corresponding sites downstream of the leftward ie1 promoter in each of these plasmids. This yielded key intermediate plasmids designated pDIE1DsRed1-TOPO.4, pDIE1ECFP-TOPO.4, and pDIE1EYFP-TOPO.4, each of which included the ie1-hr5-ie1 regulatory element for constitutive expression of heterologous gene pairs, unique restriction sites downstream of both promoters for insertion of the heterologous genes of interest, and a fluorescent eye color marker for the identification of transgenic offspring.
For the purposes of a project that will be described elsewhere, we subsequently inserted six heterologous mammalian genes encoding enzymes involved in protein N-glycosylation into the three key intermediate plasmids described above (Fig. 3). A human N-acetylglucosaminyltransferase II (GnTII; ) coding sequence was PCR amplified using pHG30  as the template and humanGlcNAcTIIsense plus humanGlcNAcTIIanti (Table1) as the primers. Similarly, the sequence encoding bovine β1,4-galactosyltransferase (GalT; ) was amplified using pBSKS-β4GalT as the template and bovineβ 4GalTsense plus bovineβ4GalTanti (Table 1) as the primers, the sequence encoding a rat α2,6-sialyltransferase (ST6GalI; ) was amplified using pIE1HR3ST6Δcys  as the template and ratST6sense plus ratST6anti (Table 1) as the primers, the sequence encoding a mouse α2,3-sialyltransferase (ST3GalIII; [27, 28]) was amplified using pST3GalIII  as the template and mouseST3sense plus mouseST3anti (Table 1) as the primers, the sequence encoding mouse sialic acid synthase (SAS; ) was amplified using p64KDIE1TV1/SAS/CMP-SAS  as the template and mouseSASsense plus mouseSASanti as the primers, and the sequence encoding mouse CMP-sialic acid synthetase (CMP-SAS;) was amplified using p64KDIE1TV1/SAS/CMP-SAS  as the template and mouseCMP.SASsense plus mouseCMP.SASanti (Table 1) as the primers. Except for ST3GalIII, each amplification product was cloned into pCRBluntII-TOPO (Invitrogen) and error-free clones were identified by DNA sequencing. The ST3GalIII product had two single nucleotide substitutions at amino acid positions 80 (phenylalanine to serine) and 294 (glycine to cysteine), which were corrected by site-directed mutagenesis before subcloning, and the corrected amplification product was designated ST3.3m. Subsequently, PmeI-SpeI fragments encoding GnTII, ST6GalI, or SAS (designated as "Glyco-A" genes in Fig. 3) were inserted at the corresponding sites downstream of the leftward ie1 promoters and NruI-NotI fragments encoding GalT, ST3.3m, or CMP.SAS ("Glyco-B" genes in Fig. 3) were inserted at the corresponding sites located downstream of the rightward ie1 promoters in the relevant intermediate plasmids. This yielded three derivatives termed pDIE1-GnTII/GalT-DsRed1-TOPO.4, pDIE1-ST6.1/ST3.3-ECFP-TOPO.4, and pDIE1-SAS/CMP.SAS-EYFP-TOPO.4. Finally, the expression cassettes were excised from these derivatives with XbaI and each was individually subcloned into the corresponding site of the piggyBac vector, pXLBacII . This final step yielded six piggyBac vectors, designated pXLBacII-GnTII/GalT-DsRed1, pXLBacII-ST6.1/ST3.3-ECFP, and pXLBacII-SAS/CMP.SAS-EYFP, which contained the three different constitutive expression cassettes in each of the two different orientations. The three vectors containing the expression cassettes with the "Glyco-A" genes facing rightward (pXLBacII-GalT/GnTII-DsRed1.A cl 57, pXLBacII-ST3.3/ST6.1-ECFP.A cl 29, and pXLBacII-CMP.SAS/SAS-EYFP.A cl 42) were designated as vector "Set 1" and those containing these same genes facing leftward (pXLBacII-GnTII/GalT-DsRed1.B cl 1, pXLBacII-ST6.1/ST3.3-ECFP.B cl 21, and pXLBacII-SAS/CMP.SAS-EYFP.B cl 1) were designated as vector "Set 2" (Fig. 3).
Construction of piggyBac vectors for inducible expression of heterologous gene pairs
A transcriptional control element consisting of back-to-back minimal cytomegalovirus immediate early promoters (P CMVmin ) separated by a tetracycline-inducible operator (TetO7; ) was constructed in several steps (Fig. 4). First, copies of the P CMVmin element and the fused TetO7-P CMVmin element were individually PCR amplified using pTRE2hyg-luc (BD Biosciences, Palo Alto, CA) as the template and HpaCMV plus NruCMV or HpaTetO plus PmeCMV as the primers (Table 1). Each of the resulting DNA fragments was cloned into pCR2.1-TOPO (Invitrogen) and error-free clones identified by restriction mapping and DNA sequencing were designated pCR2.1-HpaCMVNru and pCR2.1-PmeTetO, respectively. Subsequently, the P CMVmin fragment was excised from pCR2.1-HpaCMVNru with HpaI and BamHI and subcloned into the corresponding sites of pCR2.1-PmeTetO to produce pCR2.1-NruTetOPme, which contained the fully assembled control element consisting of back-to-back copies of the CMV promoter separated by the tetracycline-inducible operator. This "P CMVmin -TetO7-P CMVmin " regulatory element was then excised from pCR2.1-NruTetOPme with NruI and PmeI and used to replace the "ie1-hr5-ie1" control element in a subset of the dual constitutive piggyBac vectors from Sets 1 and 2, which were described above. This yielded two new sets of piggyBac vectors (Sets 3 and 4), which were designated pXLBacII-TetO1.GalT/GnTII-DsRed.A cl 3, pXLBacII-TetO1.CMP-SAS/SAS-EYFP.A cl 35, pXLBacII-TetO1.GnTII/GalT-DsRed.B cl 18, and pXLBacII-TetO1.ST6.1/ST3.3-ECFP.B cl 30. These vectors encoded the indicated heterologous gene pairs under the control of the tetracycline inducible transcriptional control element, as well as the various fluorescent eye color markers described above.
Construction of BGHpolyA-modified piggyBac vectors
Two of the piggyBac vectors in Set 1 described above were further modified by the insertion of an additional BGHpolyA signal downstream of the 5'-TRD. A DNA fragment containing the BGH polyA signal was excised from pBGHpolyA-TOPO.1 (Fig. 1B) with ApaI and BamHI, the ends were repaired with Klenow, and the resulting blunt ended DNA fragment was inserted into the Klenow-repaired BglII site in the internal domain in the 5'-TRDs of pXLBacII-GalT/GnTII-DsRed1.A cl57 and pXLBacII-TetO1.GalT/GnTII-DsRed1.A cl 3 (Fig. 4 and Fig. 6, top). The two desired BGHpolyA-modified vectors, which had the additional BGHpolyA signal oriented in the same direction as the remnant piggyBac promoter element in the 5'-TRD (vectors E and F in Fig. 6, top), were identified by colony PCR with the primer pair BGHpolyASeq1 plus BGHsense (Table 1) and DNA sequencing. These two new vectors were designated pXLBacII-GalT/GnTII-DsRed1.BGH.A cl 2 and pXLBacII-TetO1.GalT/GnTII-DsRed1.BGH.A cl 20.
Construction of piggyBac vectors lacking the transposase promoter
Two additional piggyBac vectors lacking the piggyBac transcription start region (TSR) in the 5'-TRD [4, 18] were constructed by digesting pXLBacII with HindIII and SphI to delete nucleotides 1146-1429, repairing the ends with Klenow, and religating to produce a derivative designated pXLBacIIΔTSR. Subsequently, the constitutive GnTII/GalT expression cassette was inserted at a unique XbaI site and clones containing the insert in either orientation were identified by restriction mapping and designated pXLBacIIΔTSR-GalT/GnTII/-DsRed1.A cl 16 and pXLBacIIΔTSR-GnTII/GalT-DsRed1.B cl 13.
Construction of 3'-TRDID-modified piggyBac vectors
The final piggyBac vectors constructed for this study were designed to have an additional copy of a highly AT-rich (83% A+T), putative transcriptional activator derived from the piggyBac 3' internal domain [4, 18]. A DNA fragment containing nucleotides 789-986 of pXLBacII, which contained this putative transcriptional activator, was produced by PCR with pXLBacII as the template and 5'LTRactivator plus 3'LTRactivator as the primers (Table 1). The resulting PCR fragment was cloned into the Klenow-repaired BglII site downstream of the leftward-facing GalT gene in pXLBacII-GalT/GnTII-DsRed1.A cl 57. Derivatives containing the insert in either the forward or reverse orientation, with respect to the GalT gene, were identified by restriction mapping and DNA sequencing and designated pXLBacII-GalT/GnTII-DsRed1.LTR.F cl 16 and pXLBacII-GalT/GnTII-DsRed1.LTR.R cl 25, respectively. Analogous derivatives of the inducible piggyBac vectors were produced by inserting the blunt-ended DNA fragment containing the putative transcriptional activator sequence into the Klenow-repaired BglII sites of pXLBacII-TetO1.GalT/GnTII-DsRed1.A cl 3 and identifying clones containing the insert in either the forward or reverse orientation with respect to the GalT gene, which were designated pXLBacII-TetO1.GalT/GnTII-DsRed1.LTR.F cl 23 and pXLBacII-TetO1.GalT/GnTII-DsRed1.LTR.R cl 24, respectively. We also cloned this same putative transcriptional activator fragment in the forward orientation, with respect to the leftward-facing CMP-SAS and ST6GalI genes, using the Klenow-repaired BglII sites of pXLBacII-CMP-SAS/SAS-EYFP.A cl 42 and pXLBacII-TetO1.CMP-SAS/SAS-EYFP.A cl 35 and the Klenow-repaired SalI sites of pXLBacII-ST6.1/ST3.3-ECFP.B cl 1 and pXLBacII-TetO1.ST6.1/ST3.3-ECFP.A cl30, respectively. This yielded pXLBacII-ST6.1/ST3.3-ECFP.LTR.F cl 10, pXLBacII-TetO1.ST6.1/ST3.3-ECFP.LTR.F cl 11, pXLBacII-CMP-SAS/SAS-EYFP.LTR-F cl 25, and pXLBacII-TetO1.CMP-SAS/SAS-EYFP.LTR-F cl 12, which completed the set of 3'-TRD-modified piggyBac vectors required for our studies. The sequence of the newly inserted putative activator fragment was directly confirmed by DNA sequencing of each of the 3'-TRDID-modified piggyBac vectors.
Cultures containing 2 × 106 Sf9 cells were transfected with 10 μg of the relevant plasmid DNA(s) using a modified calcium phosphate precipitation method . At 24 h post-transfection, the cells were washed once with ice-cold Tris-buffered saline (TBS; 50 mM Tris.Cl, pH7.5, and 150 mM NaCl) and once with the buffer to be used for the relevant glycosyltransferase activity assay. The cells were then extracted with the same glycosyltransferase buffer supplemented with 1% (v/v) Triton X-100 (Sigma-Aldrich, St. Louis, MO) and the extracts were frozen prior to being used for the glycosyltransferase assays. The tetracycline-inducible piggyBac vectors were assayed using a slightly different method in which Sf9 cells were co-transfected with the vector of interest in the presence of a helper plasmid, pBSK.Hr5IE1.rtTAM2.SV40-1.1k.ITR-3xP3-EGFP, which encodes the mutant repressor needed to induce transcription in the presence of tetracycline. At 12 h post-transfection, these cell cultures were treated with fresh growth medium containing 1.0 μg/mL doxycycline (BD Biosciences) and they were extracted 24 h later.
The GnTII, GalT, ST6, and ST3 enzyme activity assays were performed as described previously [16, 26, 28, 34]. The cell extraction and enzyme assay buffers used for these experiments were GnTII buffer [100 mM MES, pH 6.1, 100 mM NaCl and 1% (v/v) Triton X-100], GalT buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 20 mM MnCl2, and 0.5% Nonidet P-40), ST6 buffer (50 mM Na2HPO4, pH 7.5, 100 mM NaCl, 10 mM MgCl2, and 1.5% Triton CF-54), and ST3 buffer (100 mM sodium cacodylate, pH 6.4, 10 mM MgCl2, 2 mM CaCl2, and 1.5% Triton CF-54). Cells were washed with these buffers minus the detergents and extracted with these buffers plus the detergents, as described above, and the extracts were frozen at -85°C. Prior to performing the assays, the cell extracts were thawed, clarified at 1,000 × g for 5 min at 4°C in a microcentrifuge, and total protein concentrations were determined using a commercial bicinchoninic acid assay (Pierce, Rockford, IL) with BSA as the standard. Duplicate samples of each extract, containing 100 μg of total protein, were then incubated at 37°C for 1 h with donor and acceptor substrates in the appropriate buffers and supplements. The final GnTII assay reaction contained 67 mM MES (pH 6.1), 67 mM NaCl, 15 mM MnCl2, 6.7 mM AMP, 133 mM N-acetylglucosamine, 0.0833 mM Manα1,6(GlcNAcβ1,2Manα1,3)-Manβ-octyl (Toronto Research Chemicals, Ontario, Canada), and 0.9 μCi of uridine diphosphate [6-3H]-N-acetylglucosamine (60 Ci/mmol; American Radiolabeled Chemicals, Inc., St. Louis, MO). The final GalT assay reaction contained 0.3 μCi of uridine diphosphate [6-3H]-galactose (9.1 Ci/mmol; American Radiolabeled Chemicals) and 830 ug/mL of ovalbumin (Sigma-Aldrich) in the GalT buffer described above. The final ST6GalI and ST3GalIII assay reactions contained 0.3 μCi of cytidine 5'-monophosphate [6-14C] sialic acid (20 Ci/mmol; American Radiolabeled Chemicals) and 310 ug/mL of asialofetuin (Sigma-Aldrich). After the 1 h incubation period, each reaction was quenched by dilution with ice cold water and radiolabeled GnTII products were collected by reverse phase chromatography with SepPak C18 cartridges (Millipore, Bedford, MA), while radiolabeled GalT, ST6GalI, and ST3GalIII products were collected by TCA precipitation onto Whatman GF/D glass microfibre filters (Whatman Inc., Florham Park, N.J.). Following elution, the amounts of radioactivity transferred to each donor substrate in duplicate reactions were measured with a Model LS-6500 liquid scintillation spectrometer (Beckman-Coulter Instruments, Palo Alto, CA), the results were averaged, and the average values were converted to the average fmol of donor substrate transferred/μg total protein/h using the specific radioactivities of the donor substrates.
Sialic acid and CMP-sialic acid assays
SAS and CMP-SAS activities were determined by measuring sialic acid and CMP-sialic acid levels in transfected cell lysates, as described previously [35, 36]. Briefly, Sf9 cells were pre-incubated for 12 h with growth medium containing 10 mM N-acetylmannosamine with or without 1.0 μg/mL doxycycline. The cells were then transfected with the relevant plasmid DNAs, incubated for another 24 h, rinsed twice with ice-cold TBS buffer, and lysed in cold TBAS buffer (0.2 M Tris. pH 9.0, 0.2 mM DTT, 20 mM MgCl2, 1% Triton X-100). The lysates were clarified, total protein concentrations were determined as described above, and duplicate assays were performed with samples containing 1.0 mg of total protein. For the CMP-sialic acid determinations, the cell lysate was pre-treated with 50 μL of 1.6 M NaBH4 to reduce the free sialic acid and then with 55 μL of concentrated H3PO4 to destroy the excess NaBH4 and hydrolyze the CMP-sialic acid. The released sialic acid was oxidized by adding 50 μL of 0.2 M NaIO4 and the excess NaIO4 was subsequently destroyed by adding 0.4 mL of 4% NaAsO4 in 0.5 M HCl. After vigorous vortexing to eliminate the brown color, 2 mL of 0.1 M 2-thiobarbituric acid (adjusted to pH 9.0 with NaOH) were added and the reaction mixtures were incubated in a 100°C waterbath for 7.5 min to generate the pink chromophore, which was extracted overnight at room temperature with 4 mL of n-butanol containing 0.6 N HCl. Finally, the organic phase was collected, absorbance was measured at 532 nm, 549 nm, and 562 nm, and nmol CMP-sialic acid was calculated using a standard conversion factor (21 × OD549 nm - 7.58 × OD532 nm) × 4.0 . The method used to measure total sialic acid content was the same as described above except the cell lysates were not pre-treated with NaBH4. The results were expressed as sialic acid or CMP-sialic acid content/μg total protein.
Isolation and analysis of transgenic insects
The representative piggyBac vectors used to produce transgenic insects were pXLBacII-CMP-SAS/SAS-EYFP.LTR-F cl 25, which encodes CMP-SAS and SAS under the control of the ie1-hr5-ie1 dual constitutive transcriptional control element, and pXLBacII-TetO1.GalT/GnTII-DsRed.A cl 3, which encodes GalT and GnTII under the control of the P CMVmin -TetO7-P CMVmin dual tetracycline-inducible transcriptional element. Drosophila strains were reared under standard laboratory conditions . D. melanogaster w1118 white eye pre-blastoderm embryos were microinjected as described previously, except there was no dechorionation step . pXLBacII-CMP-SAS/SAS-EYFP.LTR-F cl 25 was injected at a concentration of 0.5 ug/uL together with 0.3 ug/uL of phspBac, which encodes the piggyBac transposase, while pXLBacII-TetO1.GalT/GnTII-DsRed.A cl 3 was injected at a concentration of 0.6 ug/uL together with 0.4 ug/uL of phspBac. To produce a fly line encoding the transcription factor needed for tetracycline induction, pBS.rtTAM2-EGFP was co-injected together with phspBac at equal concentrations of 0.4 ug/uL. One day later, all microinjected embryos were subjected to a one hour heat shock at 37°C to induce expression of the piggyBac transposase, and they were subsequently reared at 28°C. Emerging adults were individually mated with w1118 flies, and their progeny were screened for fluorescent eye color as adults using an Olympus SZX12 fluorescent microscope equipped with YFP and RED filter sets. Positive adults were individually crossed with the w1118 flies and subsequent generations were produced to establish each separate, homozygous transgenic fly line. Constitutive expression of SAS and CMP-SAS was examined by mating individual adults from the relevant homozygous fly lines, allowing the females to lay eggs on diet with or without 10 mM N-acetylmannosamine, and rearing the hatched larvae through fourth instar on the same diets. These larvae were then homogenized in ice-cold TBA buffer and the homogenates were clarified and used to measure total protein concentrations and sialic acid and CMP-sialic acid contents, as described above. To produce transgenic fly lines capable of inducibly expressing GnTII and GalT, homozygous adults encoding the rtTAM2 transcription factor were mated with homozygous adults encoding GnTII and GalT under the control of the dual tet-inducible CMV promoter. Progeny were screened for expression of both EGFP and DsRed and then single males and single virgin females from each cross were mated. The females were placed into vials containing diet with or without 50 ug/mL of doxycycline, allowed to lay eggs, and the hatched larvae were maintained on the same diet for ten days, homogenized in GnTII or GalT assay buffer, and the homogenates were clarified and used to measure total protein concentrations and GnTII and GalT activity levels, as described above.
We acknowledge the contributions of Nikolai van Beek of Chesapeake-PERL, Inc. (Savage, MD) to the original conception and design of the parent project from which this work was derived. We also acknowledge Drs. Harry Schachter (University of Toronto), Joel Shaper (Johns Hopkins University), Jim Paulson (The Scripps Research Institute), and Shuichi Tsuji (RIKEN), for kindly providing plasmids encoding GnTII, GalT, ST6GalI, and ST3GalIII, respectively, for this work. We gratefully acknowledge financial support from the National Institutes of Standards and Technology through an Advanced Technology Program grant to Chesapeake-PERL, Inc., and from the National Institutes of Health through the RO1 mechanism (grants GM49374 to DLJ and AI48561 to MJF).
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