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ANIMAL GENETICS |
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, People’s Republic of China
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Key Words: phosphorus pollution control • phytase • pig parotid secretory protein gene • transgenic mouse
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). Although phytases can release phosphate from phytate, monogastric animals have little phytase activity in their gastrointestinal tracts; the phytate in the meal therefore passes through the tract and is excreted directly into the manure, which can cause environmental pollution with severe biological consequences. The projected growth of the livestock industry is expected to accelerate such problems. Phytase can hydrolyze phytate, releasing inorganic phosphate; therefore, the use of phytase in farm animals is the choice for controlling phosphate pollution. Phytase as a feed additive is effective. Alternatively, production of phytase can be achieved by gene transfer or transgenic technologies. Such endogenous phytase could increase the bioavailability of plant phytate and in turn lead to reduced phosphorus output.
Golovan and coworkers tested the feasibility of using the promoter of the mouse salivary gland-specific parotid secretory protein (PSP) gene, an abundant protein in mouse parotid glands, to express the phytase transgene in the saliva of transgenic mice and pigs (Madsen and Hjorth, 1985; Golovan et al., 2001a,b). Their results demonstrated that the introduction of salivary phytase transgenes into monogastric farm animals was an effective way to control phosphorus pollution.
The pig is the major livestock species in China and it would be interesting to see if a phytase transgene could be effectively expressed in pigs using the promoter of pig endogenous PSP gene, which is specifically expressed at high levels in salivary glands (Yin et al., 2004). Until now, however, the pig PSP gene promoter has not been available. In this study, we cloned the upstream promoter region of the pig PSP gene and used it to express bacterial phytase in transgenic mouse models.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Escherichia coli strains DH5
and DH10B were used for plasmid construction and grown in Luria-Bertani medium supplemented with antibiotics. Restriction endonucleases and ligase were purchased from New England Biolabs (Boston, MA); Taq and Taq Plus polymerase from Takara BIO Inc. (Tokyo, Japan). Plasmid preparation and gel extraction of DNA fragments were performed using QIA spin mediprep kit (Qiagen, Valancia, CA) and the Geneclean III kit (BIO101 Inc., Vista, CA).
Bacterial Artificial Chromosome Hybridization
Bacterial artificial chromosome (BAC) hybridization was carried out to screen the fragment containing the pig PSP gene upstream region, which is from the pig ear, using a nonradioactive, digoxigenin-labeled system. A digoxigenin-labeled 1-kb probe, which was amplified from the BAC with the forward 5'-AATCTCCGTGGATGGCTC-3' and reverse 5'-ATGAGCAGACCGCACAAG-3' primer set, according to the partial pig PSP sequence, GenBank accession no: AY197556, was used for BAC hybridization. The BAC DNA was digested with HindIII, BglII, SacI, SpeI, and XbaI, fractionated in a 0.8% agarose gel, and transferred to a nylon membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK). All detailed hybridization manipulations were as described in a standard protocol (Sambrook et al., 1989).
Validation of Genome Continuity of the Upstream Promoter Region
Through the BAC hybridization, a 10-kb fragment was selected, cloned, and sequenced. The entire pig PSP upstream promoter region (10 kb) was divided into 5 parts, which were named S1, S2, S3, S4, and S5, respectively, from initiation codon to the farthest upstream end of the cloned promoter region, and amplified from pig genomic DNA, which comes from the pig ear, and sequenced. The primers are listed in Table 1.
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) and the first 8 amino acids of the mature phytase gene (CAG AGT GAG CCG GAG CTG AAG CTT) was synthesized, in which a HindIII site (underlined) was incorporated (a silent mutation). The sequence was ligated into the vector pMD-T18 to generate the recombinant plasmid pSP/T. The HindIII sites allow for the fusion of the phytase gene to the signal peptide sequence of the pig PSP gene.
The phytase gene (appA) was cloned from E. coli K12 according to the appA sequence database deposited by Dassa et al. (1990). Total DNA from E. coli K12 was used for cloning of the appA gene as described by Davis et al. (1980) and a 1.2-kb appA without its signal peptide was amplified with the forward primer (5'-TGAAGCTTGAAAGTGTGGTGAT-3') containing a HindIII site (underlined) and the reverse primer (5'-ATGGCGCGCCTTACAAACTGCACGCCGGTAT-3') containing an AscI site (underlined) based on the appA gene sequence from E. coli strain K12 (GenBank M58708).
These appA sequences were cloned into the vector pMD-T18 (Takara BIO Inc.). Following ligation, E. coli strain DH5 was transformed using Gene Pulser (BioRad, Hercules, CA) and the desired recombinant clone pAPPA/T was sequenced 3 times from both strands. The plasmid pAPPA/T was digested with HindIII and AscI, and then the appA fragment was gel-purified and ligated into the equivalent sites of recombinant plasmid pSP/T appropriately predigested with the same restriction enzymes to generate the plasmid pSP/APPA. The identities of recombinant plasmids were verified by restriction analysis and DNA sequencing of the cloning junctions using a PRISM dye-terminator cycle sequencing kit (Applied Biosystems, Foster City, CA). Sequencing reactions were analyzed in our laboratory.
The 10-kb fragment containing the pig PSP upstream promoter region was obtained through BAC hybridization, and subcloned into the vector pGEM-3ZF, which was linearized by BamHI, creating a compatible sticky end to BglII. The resultant plasmid pPSP/3ZF was then sequenced. For construction of the phytase transgene, the 10-kb fragment was amplified by PCR from the recombinant plasmid pPSP/3ZF using a forward primer (5'-ATGCGGCCGCGAGCTCTCCGGATCTGGGCAAAGT-3') containing a NotI site (underlined) and a reverse primer (5'-ATGGCGCGCCTTGTCCCGAAACCTGGAACCATCATG-3') containing an AscI site (underlined), then inserted into pCR-XL-TOP vector (In-vitrogen, Carlsbad, CA), resulting in plasmid pPSP/TOP.
The 280-bp fragment of the bovine growth hormone polyadenylation signal region was amplified from plasmid pIRESneo (provided by our laboratory) with a forward primer (5'-ATGGCGCGCCGATCAATTCTCTAGAGCTCG-3') containing an AscI site (underlined) and a reverse primer (5'-GCAGATCTCAGCTGGTTCTTTCCGCCT-3') containing a BglII site (underlined). The PCR product was cloned into the vector pGEM-T, and the resultant plasmid, pBGH/T, was then sequenced. The pig PSP upstream promoter region was excised with AscI and NotI from pPSP/TOP and subcloned into the same sites in pBGH/T digested with same enzymes, to generate the pPSP/BGH plasmid (NotI site in original vector).
The plasmid pPSP/BGH was linearized with AscI and served as a vector. The pSP/APPA was digested by AscI and a 1.4-kb fragment containing pig PSP signal peptide and appA was recovered and then ligated into the AscI site in pPSP/BGH. This produced the final transgene pPAB (PSP/APPA/BGH), which was sequenced by our laboratory.
A 2.5-kb pig PSP downstream sequence, which contained the polyadenylation signal, was obtained by primer extension with BAC DNA as template. The 2.5-kb fragment was amplified by PCR from pig BAC using primers (forward: 5'-AGGCGCGCCCTGAATTCTCTTGTACCATCTTC-3', underlined AscI site; and reverse: 5'-CAGATCTAACTTACCTAAGACTGACATCGG-3', underlined BglII site) that insert an AscI site at the 5' end and a BglII site at the 3' end of the fragment. This AscI/BglII fragment was then inserted into pPAB (PSP/APPA/BGH), and predigested with the same endonucleases to produce pPSP/PDR.
A linear DNA fragment of 13 kb containing the upstream promoter and downstream region of pig PSP was obtained by digestion of plasmid pPSP/PDR with AscI. The second plasmid pPSP (PSP/APPA/PDR) containing the pig PSP upstream and downstream as well as signal peptide/appA was constructed by insertion of the signal peptide/appA fragment, from plasmid pSP/APPA digested with AscI, into the same endonuclease-digested pPSP/PDR vector. The final construct pPSP (PSP/APPA/PDR) contains the pig PSP signal peptide/appA and pig PSP 3' flank region, all under the control of the pig PSP upstream promoter region.
Transgene Purification, Quantification, and Pronuclear Microinjection
The linear DNA fragment containing the PSP promoter, signal peptide, and phytase gene, as well as the 3' polyadenylation signal, were obtained by digestion of the 2 final constructs, pPAB and pPSP, with NotI/BglII and subsequent purification from an agarose gel slice by electroelution, followed by phenol/chloroform extraction, dialysis in Tris-EDTA buffer [10 mMol/L NaCl, 10 mMol/L Tris.Cl (pH 8.0), 1 mMol/L EDTA (pH 8.0)] and ethanol precipitation. Finally, DNA was resuspended in microinjection buffer [10 mMol/L Tris.Cl (pH 7.4), 0.1 mMol/L EDTA] at a concentration of 20 ng/µL, and stored at –20°C.
The injection of transgene DNA was carried out according to the procedure described by Hogan et al. (1986).
Southern Blotting Analysis
Mice genomic DNA was isolated from the tails (72 transgenic founders and 2 controls) by phenol-chloroform extraction. Ten micrograms of DNA was digested with XbaI and PvuII, fractionated in a 0.7% agarose gel, and transferred to a nylon membrane (Amersham Pharmacia Biotech). The membrane was hybridized with 32P-labeled appA DNA (1.2-kb) probe. Hybridization and washing were performed according to the procedures of Sambrook et al. (1989). Prehybridization was carried out at 65°C for 6 h, hybridization for 24 h at 65°C, then washed the membrane with 2x SSC, 0.5% SDS at 65°C twice, and 2x SSC, 0.1% SDS once. For the second mouse model, the genomic DNA was digested with BamHI.
Saliva Collection
Among the stimulants, pilocarpine has been the favored drug compared with a wide variety of pharmacological salivary secretagogues. Mice aged 2 mo were given an intraperitoneal injection of pilocarpine (2.5 µg/g of BW; Koller et al., 1992). Salivation in the experimental mice was monitored continuously by blotting the mouth with discs of filter paper. Once salivation became visible, the salivary fluid was aspirated using a micropipette fitted with a bent disposable tip and collected into Eppendorf tubes, which were kept on ice throughout the procedure.
At the end of or during the collection procedure, the Eppendorf tubes were weighed to estimate the amount of collected saliva by assuming a specific gravity of 1.00 g/mL. In the case of multiple collection procedures, salivary stimulation in the same mouse was scheduled at 48-h intervals. Between 200 and 300 µL of saliva was collected from 2-mo-old mice as described above and stored at –80°C.
Phytase Activity Analysis
Phytase activity in saliva was measured in an assay mixture containing 0.5% phytic acid and 200 mM sodium acetate (pH 5.0). After 15 min of incubation at 37°C, the reaction was terminated by adding an equal volume of 15% trichloroacetic acid. The liberated phosphate ions were quantified by mixing 100 µL of the assay mixture with 900 µL of H2O and 1 mL of 0.6 M H2SO4, 2% ascorbic acid, and 0.5% ammonium molybdate. Absorbance at 820 nm was measured after 20 min of incubation at 50°C. Standard solutions of sodium phosphate were used for calibration. One unit of phytase activity was defined as the amount of activity that liberates one micromole of phosphate per minute at 37°C.
Collection of Fecal Materials and Analysis for Phytate
The transgenic and control mice were individually caged and fed with rodent chow containing 0.27% (wt/wt) phytate and 0.4% (wt/wt) nonphytate phosphorus. Fecal samples were collected once daily for 2 wk. Samples were placed into sterile tubes and dried immediately.
The standard phosphorus concentration curve was established before the determination began. Dried feces were analyzed according to the quantitative trichloroacetic acid colorimetric method. Phytate was extracted from 3 to 6 g of dried feces with 3% trichloroacetic acid on a shaker platform for 30 min, fractionated, and then precipitated by ferric trichloride according to the method described by Wheeler and Ferrel (1971) with modification. Colorimetric assay was carried out at 420 nm.
Statistical Analyses
Sigmastat (Systat Software Inc., Point Richmond, CA) was used in all statistical evaluations. The 2-wk total fecal phytate per mouse was used to estimate the variation. The Mann-Whitney rank sum test, a nonparametric procedure that does not require assuming normality or equal variance, was applied to test for a difference between the 2 groups (control and transgenic) that is greater than that which can be attributed to random sampling variation.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). Initially, we tried to determine the pig PSP promoter by aligning the known mRNA with pig genomic sequences. This approach has been reported to have over 80% accuracy (Qiu, 2003; Trinklein et al., 2003). However, largely due to the limited sequence of the transcript available, the transcription initiation site and the further upstream sequence of the pig PSP gene could not be identified. We then applied BAC hybridization to obtain the upstream promoter region of the pig PSP gene. The same pig BAC clone was digested with 5 different restriction enzymes, HindIII, BglII, SacI, SpeI, and XbaI. These enzymes were selected because their digestion products would contain the small part of the known 5' flanking region. The digestion fragments were hybridized with the probe labeled with digoxigenin and one positive fragment of 10 kb was selected, cloned, and sequenced (GenBank accession no. AY197556).
PromoterInspector software was used for searching the candidate promoter of pig PSP gene (Bucher, 1990; Scherf et al., 2000). A TATA box was located 40 bp upstream to the exon 1 (untranslated region). Further comparison of upstream regions between the pig and mouse PSP gene identified a conserved TATA box and untranslated exon 1 and other common regulatory elements including AP, C/EBP, OTF1, Oct-1, and GC boxes (Figure 1). These elements are shared in the pig and mouse PSP gene, indicating a similar regulatory mechanism of the PSP gene family (Svendsen et al., 2001).
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), with at least 100-bp overlapping junctions to each other. The PCR products were 1.96, 1.84, 2.5, 1.5, and 1.6 kb in length, respectively, and were from the initiation codon to the farthest part of the fragment, respectively (Figure 2). Subsequent sequencing analysis of the PCR products demonstrated that the BAC clone and pig PSP cDNA contained the correct sequences (Yin et al., 2004).
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. The model was designed with the upstream promoter of the pig PSP gene, the bacterial phytase gene, and the bovine growth hormone polyadenylation signal, to evaluate the possible regulatory efficiency of the pig PSP upstream promoter.
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The intactness, integration, and copy numbers of the inserted transgenes were established by Southern blotting with a 1.2-kb -P32-phytase gene probe. The 3.5-kb fragments, comprising the 2-kb upstream promoter of the pig PSP gene, the bacterial phytase (appA) gene, and the bovine growth hormone polyadenylation signal, were derived from digestion of pPAB plasmids with PvuII and XbaI, and used to assess the intactness and copy number of inserted transgenes. The results showed that the transgene was integrated into the genome, with the copy number of the transgene ranging from 1 (animal no. 70) to 100 copies.
To evaluate the effect of the 3' polyadenylation signal of the pig PSP gene on transgene expression, the second transgenic model was generated by replacing the bovine growth hormone 3' terminator with the 2.5-kb pig PSP polyadenylation signal, which was obtained by direct sequencing of the pig BAC (GenBank no. AY197556). The 13.8-kb linear transgene pPSP was produced with digestion by NotI and BglII (see map in Figure 3). After microinjection, 7 transgenic founder (G0) mice (4 males and 3 females) were identified by PCR and Southern blotting with the same 1.2-kb phytase gene as the probe. The copy number of the transgene ranged from 1 to >15, with single-copy inserts in transgenic mice nos. 14, 34, and 51, and more than 15 copies in mouse no. 45.
Functional Activity of Phytase in the Transgenic Mice
Saliva samples of pPAB transgenic and control mice (6 transgenic mice and 4 control mice) were collected and examined for phytase expression. Phytase was detected in the saliva at concentrations up to 0.24 ± 0.07 U/mL for the first transgenic mouse models (pPAB) and up to 0.33 ± 0.01 U/mL for the second model (pPSP); no phytase was detected in the saliva of control mice.
The phytase activity present in the saliva of different transgenic founder lines differed considerably despite a similar copy number of the transgene (control 0.06 ± 0.03; first transgenic line 0.24 ± 0.07; second transgenic line 0.33 ± 0.01). This is likely to be the positional effect of transgene insertion, a phenomenon commonly observed in transgenic mice (Allen et al., 1988; Al-Shawi et al., 1990), which might play an important role for the activation of the transgene. The expression of phytase transgenes in mouse saliva indicates that the 10-kb PSP gene upstream promoter region contains the essential elements required for achieving salivary-specific expression.
To further determine the effect of the expressed phytase on the transgenic mice, the nutritional potential of salivary phytase was investigated by analyzing the phytate concentration in fecal dry matter from the same transgenic and nontransgenic mice with a diet containing 40% of total phosphorus as phytate. Consistent with the detectable expression of phytase in the saliva, we detected a significant difference in fecal phytate concentration between transgenic and nontransgenic mice for both transgenic mouse models pPAB and pPSP (Figure 4).
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0.001) compared with that in nontransgenic mice (0.70 ± 0.02%). Transgenic pPSP mice produced feces with a phytate concentration of 0.60 ± 0.02%. This represents a reduction of 12.5% (P < 0.05) in phytate concentration compared with nontransgenic animals (0.69 ± 0.04%) under the same conditions. Compared with the first mouse model, the phytate concentration in the second model was decreased (P < 0.005). The results suggest that the pig PSP downstream region increased the expression more than the bovine hormone growth polyadenylation signal. In conclusion, we have demonstrated that the proximal PSP promoter is sufficient to drive the expression of the phytase transgene in mouse saliva. The salivary appA phytase in these mice leads to a significant reduction of fecal phytate levels. Introduction of a phytase transgene into pigs and promoting its expression with pig PSP promoter would be expected to produce a significantly larger amount of the enzyme and a decrease in fecal phytate content. This finding establishes the basic principles for the production of phytase transgenic pigs.
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Footnotes |
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2 Appreciation is expressed to Q.-L. Lu (MRC, Clinical Sci. Center, Imperial College, London, UK) for critically reviewing the manuscript.
3 Current address: Muscle Cell Biol., Med. Res. Council, Clinical Sci. Centre, Imperial College, Faculty of Med., Hammersmith Hosp., London W12 0NN, UK.
4 Corresponding author: ninglcau{at}cau.edu.cn
Received for publication April 24, 2005. Accepted for publication September 28, 2005.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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