| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Monsanto Company, Chesterfield, MO 63017 and
and
University of Kentucky, Lexington, KY 40546
4 Correspondence:
Mail Stop BB5B, 700 Chesterfield Pkwy West (phone: 636-737-5835; fax: 636-737-7662; E-mail:
james.jennings{at}monsanto.com).
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
Key Words: Biotechnology • Enzyme-Linked Immunosorbent Assay • Pigs • Polymerase Chain Reaction • Soybeans
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
The present study describes DNA and protein analytical methods optimized for high sensitivity to test for fragments of the cp4 epsps and le1 (encoding soy lectin) genes and both the intact CP4 EPSPS protein and its immunoreactive fragments in loin muscle samples from finishing swine fed a diet containing RR soybean meal.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
The swine from which tissues were obtained were from a feeding study to assess growth performance and carcass composition of pigs fed diets containing dehulled soybean meal prepared from RR and conventional soybeans (Cromwell et al., 2002). Pigs (five replications of barrows and five replications of gilts with five pigs per pen) were fed fortified corn-soybean meal diets containing 24, 19, or 14% RR or conventional soybean meal on an air-dry weight basis during grower (24 to 55 kg), early-finisher (55 to 87 kg), and late-finisher (87 to 111 kg) phases of growth, respectively. The identity of the soybeans used to formulate both the conventional and transgenic diets was confirmed by a standard sandwich ELISA for CP4 EPSPS protein prior to diet formulation. In addition, the meal used to formulate the diets was also tested by CP4 EPSPS immunoassay using a Traitcheck RUR lateral flow test (Strategic Diagnostics Inc., Newark, DE) to confirm the presence or absence of the transgenic protein in the test and control processed meals, respectively. At the termination of the experiment, the barrows were killed. A sample of the longissimus muscle was collected from two or three randomly selected barrows from each pen (12 per treatment) and placed in individual plastic bags with labels indicating each animal’s ear notch number, sample collection date and tissue type, and then immediately frozen and shipped to the laboratory on dry ice. Animal selection was based on randomly selected ear notch numbers prior to sample collection, and gilts were retained for use as breeding stock.
Loin muscle samples from 12 pigs fed diets containing RR soybean meal and 12 pigs fed diets containing conventional soybean meal were subjected to DNA and protein analyses. The ear notch numbers were used to track tissue samples, resulting in a blinded sequence of samples that was maintained throughout the subsequent analyses. Frozen tissue samples were further processed in the laboratory using aseptic techniques as described by Jennings et al. (2003). All subsampling occurred in a laboratory where no PCR products or plasmids were handled. Subsamples were stored in a -80°C freezer until analyzed.
Deoxyribonucleic Acid Analysis
Deoxyribonucleic Acid Extraction. Pork loin samples (2 to 9 g) were homogenized and treated with Proteinase K and RNase A as previously described (Jennings et al., 2003). Immediately following a 30-min incubation, 250 µL of phenol:chloroform:isoamyl alcohol (25:24:1, vol/vol/vol) was added to each 600-µL homogenate and vortex mixed. The homogenate was then centrifuged at 12,000 x g to separate the phases, and the aqueous phase was transferred to a 1.5-mL tube (previously treated with UV light to destroy any potential DNA contamination). A one-third volume of 7.5 M ammonium acetate and one volume of 100% isopropanol were added to the aqueous phase. The solution was gently mixed by inversion for approximately 5 min, and then the DNA was precipitated by centrifugation at 12,000 to 14,000 x g for 15 to 20 min. DNA pellets were washed with 70% (vol/vol) ethanol, dried under vacuum, and then resuspended in 100 µL of 1x TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). To serve as a positive control for the extraction procedure, approximately 250 pg of purified genomic DNA from RR soybeans was spiked into tissue homogenates containing 120 to 150 mg of starting tissue immediately prior to the start of the DNA extraction procedure. A negative DNA extraction control, consisting of a 600-µL aliquot of extraction buffer subjected to the extraction procedure, was also included with each set of samples. Every pork loin sample was extracted in duplicate. Each extract was analyzed by PCR for cp4 epsps and le1.
Polymerase Chain Reaction and Southern Blot Analysis.
Extreme caution was used when handling animal samples and DNA extracts so as not to contaminate them with previously amplified DNA, plasmids containing transgenes, or any other potential sources of plant and/or transgenic material (Jennings et al., 2003). Extracted DNA was quantitated using Hoefer’s DyNA Quant 200 Fluorometer or Molecular Devices’ fmax Fluorescence Microplate Reader with Hoechst dye. Prior to the initiation of these studies, a standard preparation of genomic DNA from RR soybean event 40-3-2 was extracted, purified, and characterized, and then used throughout these analyses as the reference standard for PCR. Experiments with the cp4 epsps and le1 PCR assays were conducted with this purified RR soybean genomic DNA added to pork genomic DNA extract to determine that up to 10 µg of pork DNA could be used in both assays without detectable matrix effects. Therefore, the PCR assays were conducted with 1 µg of genomic DNA in a 50-µL total reaction volume. Pipetters dedicated for PCR setup were used with sterile, aerosol-resistant tips. Each reaction was performed in a PTC-225 DNA Engine Tetrad thermocycler (MJ Research, Inc., Watertown, MA) and contained 2.5 mM MgCl2, 0.2 µM each primer, 1 µL of PCR Nucleotide MixPlus (Roche), 2 units of REDTaq DNA Polymerase (Sigma, St. Louis, MO), and 2 units of uracil-DNA glycosylase (Roche) in 1x reaction buffer. Uracil-DNA glycosylase and dUTP (instead of dTTP) were used to eliminate any amplicon contamination. The PCR cycling conditions for amplification of the cp4 epsps sequence fragment were: 1 cycle at 37°C for 20 min; 1 cycle at 95°C for 5 min; 35 cycles at 94°C for 30 s, 65°C for 30 s, 72°C for 45 s; and 1 cycle at 72°C for 5 min. PCR cycling conditions for the endogenous soybean gene, le1 (encoding soy lectin, GenBank Accession No. K00821), were: 1 cycle at 37°C for 20 min; 1 cycle at 95°C for 5 min; 35 cycles at 94°C for 30 s, 52°C for 30 s, 72°C for 1 min; and 1 cycle at 72°C for 5 min. Primers for cp4 epsps and le1 amplification are listed in Table 1
. Each set of PCR assays included both positive and negative controls. The positive controls were reactions containing 100 ng of purified RR soybean genomic DNA. Additional positive controls were the DNA extracts from tissue samples spiked with RR soybean genomic DNA as described above. Negative PCR controls did not contain any template DNA. Additional negative controls were the buffer-only extracts described above.
|
As described above, appropriate positive and negative controls were included in all of the analyses to ensure the sensitivity and reproducibility of the DNA extractions and PCR assays. In addition, all DNA extracts were analyzed by PCR for a 185-bp region of the porcine preprolactin (prl) gene (GenBank Accession No. X14068) to ensure the quality and suitability of the DNA extracts for PCR. Reaction conditions were the same as those used for cp4 epsps and le1, except omitting the uracil-DNA glycosylase and using 100 ng of DNA template instead of 1 µg. Cycling conditions for the prl assay were: 1 cycle at 94°C for 3 min; 35 cycles at 94°C for 30 s, 50°C for 30 s, 72°C for 90 s; and 1 cycle at 72°C for 5 min. Primers for prl amplification are listed in Table 1
. Products of prl amplification were visualized on an ethidium bromide-stained agarose gel. Limit of detection (LOD) studies were not conducted for the prl gene fragment because a relatively large amount of pig genomic DNA was used in each reaction. Assuming prl is a single-copy gene, then 100 ng of pig genomic DNA contains tens of thousands of copies of prl, and 1 µg of pig genomic DNA contains hundreds of thousands of copies of prl. Assays for prl ensured the quality of the DNA being tested.
Acceptance criteria for a "true positive" and "true negative" PCR result are described in Jennings et al. (2003) with the following modifications: each extract in this study was analyzed only once by each PCR assay (instead of by duplicate assays for each extract), and there was only one spiked extraction control per set of analyses (instead of spiked controls at two concentrations per set). In addition, each set of extractions and assays in this study included a buffer-only extract as described above. These changes, coupled to the testing scheme shown in Figure 1, helped to streamline the analysis process while maintaining rigorous and comprehensive controls.
|
Protein Extraction. Tissue samples were lyophilized, disrupted, extracted for protein, and then stored as previously described (Jennings et al., 2003), except that 20 volumes of extraction buffer were used for each extraction rather than 10 volumes.
Competitive Immunoassay. Analysis of tissue samples for the presence of CP4 EPSPS protein was performed using a competitive ELISA. Recombinant CP4 EPSPS protein, used in the ELISA analyses described below, was expressed in E. coli and purified from batch culture. The identity and purity of this CP4 EPSPS standard was confirmed prior to its use in these studies. CP4 EPSPS was diluted to 0.8 µg/mL in coating buffer (50 mM carbonate buffer, pH 9.6) and 100 µL per well was dispensed into 96-well microtiter plates. The CP4 EPSPS protein was immobilized to the microtiter plates by incubation overnight at 4°C. After immobilization, each well was aspirated and washed three times with PBST washing buffer [10 mM phosphate buffer, pH 7.4, 0.8% (wt/vol) NaCl, 0.05% (vol/vol) Tween-20] and then blocked with 1% (wt/vol) nonfat dry milk in PBST washing buffer overnight at 4°C. Sera from goats that were immunized with purified, recombinant CP4 EPSPS protein were purified on a CP4 EPSPS immunoaffinity column (Pierce, Rockford, IL) to obtain antibodies specific for CP4 EPSPS protein. The specificity of these antibodies was confirmed by Western blot analysis of purified CP4 EPSPS protein standard (data not shown). The CP4 EPSPS-specific, immunoaffinity purified antibodies were preincubated overnight at 4°C at a concentration of 0.05 µg/mL with either: 1) tissue sample extracts or 2) protein standards in the presence of an equivalent concentration of tissue extract. Into each well of the CP4 EPSPS protein-coated microtiter plate was transferred 100 µL of the antibody/extract mixture.
The CP4 EPSPS competitive immunoassay was initiated by incubating 100 µL of standard or test sample in each well of a plate for 1 h at 37°C. After aspiration and washing each well three times with PBST washing buffer (
300 µL/well), 100 µL of a 1:3,000 dilution of rabbit anti-goat polyclonal antibody, conjugated to horseradish peroxidase in stabilizing buffer (Stabilzyme HRP Conjugate Stabilizer, SurModics, Eden Prairie, MN), was incubated in each well of the assay plate for 1 h at 37°C. Following aspiration and washing of each well three times with PBST washing buffer (300 µL/well), 100 µL of the enzyme substrate 3,3',5,5'-tetramethylbenzidine (KPL, Gaithersburg, MD) was added per well and allowed to develop for approximately 10 min at room temperature. The enzymatic reaction was terminated by the addition of 100 µL per well of 3 M H3PO4. Immediately after stopping the enzymatic reaction, absorbance readings were determined using a dual wavelength plate reader at 450 nm with a reference wavelength of 650 nm. The concentration of CP4 EPSPS protein was determined in each sample by interpolation against a serially diluted eight-point standard curve, with values that ranged from 0.25 to 4.0 ng/mL. The standard curve was determined by a quadratic curve-fitting model (Microsoft Excel). The LOD for this assay was determined to be approximately 94 ng of CP4 EPSPS protein/g of pork loin muscle tissue (94 ppb).
Protein data from conventional soybean-fed animals were compared to data from RR soybean-fed animals using Microsoft Excel (t-test: paired two sample for means analysis). All samples were below the LOD for the assay. However, there was some variance around the level of the inhibition. Therefore, to determine if there was a statistically significant difference between the mean of the two populations, samples were analyzed using a Student’s t-test, which assumes that the means of both data sets are equal; it is also referred to as a homoscedastic t-test. The test and control populations consisted of seven samples each. For the hypothesized mean difference, a value of zero was entered to indicate that the sample means are hypothesized to be equal. The alpha value used for the test was 0.05 with a 95% confidence level. There was no statistically significant difference between the test and control sample populations at a 95% confidence level.
Intact CP4 EPSPS protein was subjected to simulated in vitro gastric conditions using a U.S. Pharmacopia (USP) method consisting of approximately 15 activity units/mL of pepsin (Sigma) in a pH 1.3 solution of simulated gastric fluid at approximately 18°C as previously described (Harrison et al., 1996). The pepsin enzyme was inactivated after 20 s by adding 100 µL of the digested sample to 200 µL of carbonate-bicarbonate buffer, pH 9.6 (Sigma). After pepsin enzyme was inactivated, one volume of 2x Laemmli buffer (Sigma) was added to the sample, and the sample was incubated in a boiling water bath for 2 min. Sample was then analyzed by SDS-PAGE on a 4 to 12% (wt/vol) gradient gel using a mini gel system (Bio-Rad, Hercules, CA). Electrophoresis was conducted at 150 V for about 1 h, and the gel was then stained with Brilliant Blue R (Sigma) and destained in 40% (vol/vol) methanol. Undigested CP4 EPSPS standard and prestained molecular weight markers (Novex MultiMark Multi-Colored Protein Standard, Invitrogen, Carlsbad, CA) were loaded onto the gel so the approximate molecular weight of the peptides could be confirmed. To demonstrate the ability of the method to detect peptide fragments of CP4 EPSPS, the partially digested protein was compared to intact CP4 EPSPS in the competitive ELISA.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
PCR followed by Southern blot hybridization was used to analyze DNA extracted from pork loin muscle samples for a 272-bp fragment of the cp4 epsps coding region and a 198-bp region of the endogenous soybean gene le1 (encoding soy lectin). Inherent to coupling sensitive PCR methods with Southern blotting is the increased risk of both false positives (i.e., a result due to contamination of the extract or reaction with template DNA) and false negatives (i.e., the presence of a PCR inhibitor in the extract). Therefore, all analyses were conducted in a sample-blinded manner following a detailed testing scheme (Figure 1) and applying a well-defined set of criteria for accepting "true positive" and "true negative" PCR results. Before a sample could be confirmed as having either detectable or undetectable levels of plant or transgenic DNA, the criteria described in Jennings et al. (2003) had to be met. First, samples were always extracted in at least duplicate and each extract was analyzed by PCR. For a Southern blot to be considered valid, both positive and negative control reactions had to produce the expected results. As a positive control for both extraction and PCR, the spiked sample had to produce a positive result on the Southern blot. As a negative control for both extraction and PCR, the buffer-only extract had to produce a negative result on the Southern blot. Lastly, to ensure that the DNA was amenable to PCR and did not contain significant inhibitors, each tissue extract was used in a PCR to amplify an endogenous gene fragment from prl. In addition, the results had to be consistent between duplicate extracts/reactions for a sample to be called negative or positive (Figure 1). As illustrated, if testing results were not consistent after three rounds of extraction and PCR, the results for that sample were termed "indeterminate."
Both assays were shown to be capable of detecting as little as 1 pg of purified genomic DNA from RR soybeans in the presence of 10 µg of swine genomic DNA (Figures 2 and 3). Routinely, however, the LOD was approximately 2.5 pg, or roughly one diploid genome equivalent of soybean DNA (Arumuganathan and Earle, 1991). Figures 2 and 3 show that it is difficult to visualize the low amounts of PCR product generated from picogram amounts of genomic template using merely ethidium bromide staining of an agarose gel. For example, PCR product from the le1 assay is not visible on a stained gel unless more than 25 pg of purified template DNA is used in the reaction and, in fact, the gene-specific, 198-bp band is much weaker than the nonspecific amplicons that arise in the presence of pork matrix (Figure 3). However, when coupled to Southern blot analysis with specific 32P-labeled probes, the presence of amplicon is unequivocal for both cp4 epsps and le1 assays (Figures 2 and 3). The sensitivity of the Southern method was so great, as measured by the specific binding of radiolabeled probe, that exposures of less than 1 h were routinely sufficient to visualize a PCR product. Blots, however, were purposefully overexposed to film (typically twice the optimal exposure) for higher sensitivity. Not only is the sensitivity of each assay increased by Southern blotting, but also the clear bands obtained on an autoradiograph alleviate some of the uncertainty inherent in the interpretation of a gel result, such as the nonspecific amplicons that are evident in the presence of pork matrix with the le1 PCR assay (Figure 3). As the number of cycles used in PCR is increased to achieve greater assay sensitivity, or when the assay is conducted in the presence of a relatively large amount of animal DNA, the possibility of producing non-specific or questionable PCR amplification products increases.
|
|
. The table shows that none of the extracted DNA from pork loin muscle samples was positive for cp4 epsps or le1 according to the testing scheme in Figure 1 and criteria described above and, in fact, all samples met the criteria for being true negative results. No results were indeterminate for any tested sample.
|
An immunoassay for CP4 EPSPS protein has been described (Lipp et al., 2000) that involves capture of the protein by immobilized antibody followed by detection of the captured protein with a second CP4 EPSPS antibody preparation (i.e., a sandwich ELISA). However, a critical component of the safety assessment of proteins introduced into agricultural biotech products approved for food and feed use is the demonstration that these introduced proteins are readily digestible in simulated gastric models (Metcalfe et al., 1996). Based on the safety assessment of CP4 EPSPS protein, it was highly unlikely that intact CP4 EPSPS protein would be found in samples of muscle tissue from pigs fed RR soybean meal. A competitive ELISA was therefore developed that had the potential to detect partially digested forms of CP4 EPSPS protein in addition to intact protein. In the competitive ELISA, incubation of CP4 EPSPS protein and its immunoreactive fragments with anti-CP4 EPSPS antibodies in solution allows a binding equilibrium to be reached between the protein in solution with the CP4 EPSPS protein immobilized to the assay plate. Fragments of partially digested CP4 EPSPS protein that can bind to anti-CP4 EPSPS antibodies will be detected in this assay configuration, whereas some of these fragments would not be detected in a sandwich assay configuration due to the requirement of at least two accessible antibody epitopes being present on the peptide. The ability of the competitive assay to detect fragments of protein was demonstrated by digesting CP4 EPSPS protein in USP simulated gastric fluid for approximately 20 s (Figure 4), and then analyzing the digested protein in both the sandwich and competitive ELISA. The sandwich ELISA demonstrated no reactivity with the digested CP4 EPSPS protein up to a concentration of 16 ng/mL (i.e., all optical densities in sample wells were less than or equal to the optical densities of buffer-only wells; data not shown). However, the competitive assay successfully detected partially digested CP4 EPSPS protein in a manner very similar to intact CP4 EPSPS (Figure 4). It is important to note, however, that fully digested CP4 EPSPS is no longer immunoreactive and would not be detectable by any ELISA. Digestion of the CP4 EPSPS protein in simulated gastric fluid for times greater than 20 s results in increased degradation of the protein. Presumably, a point of degradation would be reached where the competitive ELISA could no longer detect CP4 EPSPS protein fragments.
|
), with values that ranged from 0.25 to 4.0 ng/mL in the presence of 10% (vol/vol) pork tissue extract. To normalize for the possible impact that tissue matrix could have on binding of antibody to CP4 EPSPS protein fragments in sample extracts, the standard curve was generated in the same concentration of tissue homogenate as the test samples. The curve in Figure 5 was generated using a quadratic curve-fitting model. The LOD for this assay was determined to be approximately 94 ng of CP4 EPSPS protein/g of pork muscle tissue. There was no statistical difference (P < 0.05) in measurements between homogenates of pork loin samples derived from animals fed RR or conventional soybean meal when analyzed by the competitive immunoassay (Table 2).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
The present study was conducted to assess whether a characterized DNA detection method, highly optimized for sensitivity, could detect fragments of transgenic DNA from RR soybean meal in muscle from pigs fed this genetically enhanced product. Highly sensitive methods are needed because the amount of transgenic DNA that an animal consumes per day is very small, especially compared to the total amount of DNA consumed. For example, it has been calculated that a 600-kg dairy cow fed a diet enriched in insect-protected corn containing a Cry protein derived from Bacillus thuringiensis (Bt corn), with 40% of the diet as Bt grain and 20% as Bt corn silage, will consume about 600 mg of total plant DNA each day, with less than 3 µg being transgenic DNA (Beever and Kemp, 2000).
In the current study, relatively small DNA fragments (272 and 198 bp) of the glyphosate-tolerance transgene (cp4 epsps) and an endogenous soybean gene (le1) were not detected by PCR methods involving a relatively high level of input DNA followed by Southern blot hybridization of the amplified product. The routine LOD for the present PCR/Southern blot detection of cp4 epsps and le1 gene fragments was approximately 2.5 pg of purified RR soybean genomic DNA per reaction. The LOD for these PCR/Southern methods is essentially at the theoretical limit of the assays by detecting a single diploid genome equivalent of soybean DNA (Arumuganathan and Earle, 1991). The appropriate positive and negative DNA extraction and PCR controls, including a control reaction for an endogenous pork gene (prl), data acceptance criteria, and testing scheme, are included in the present study to characterize and verify the results.
The present results parallel data on the fate of the cp4 epsps gene in other animal species. Klotz and Einspanier (1998) showed that a 200-bp portion of the cp4 epsps coding region was not detected by PCR and Southern blot analysis in blood or milk from dairy cows fed RR soybeans. However, it was reported that a fragment of a highly abundant chloroplast gene was detectable in the white blood cells, but not in the milk of these dairy cows. A simple explanation of these results is that the chloroplast gene fragment was detected because of its significantly greater abundance in plant cells compared to the single haploid copy of the coding region for cp4 epsps in RR soybeans.
Our results also are similar to the data obtained from a study with broiler chickens that were fed RR soybean meal (Khumnirdpetch et al., 2001). In that study, real-time PCR was used to test whether a fragment of transgenic DNA was detectable in meat, skin, duodenal, or liver samples from broilers fed RR soybean meal over the entire life of the birds. The investigators found that all samples from the 7-wk study were negative for transgenic DNA.
Just as the DNA detection methods were optimized for detection of small fragments of transgenic DNA in pork loin samples, the present study also described a protein detection immunoassay method that was specifically developed to determine whether intact CP4 EPSPS protein, or immunoreactive fragments of this protein, could be detected in loin samples from pigs fed RR soybean meal. The CP4 EPSPS protein is readily degraded under simulated gastric digestion conditions, with this protein being almost totally degraded within 30 s (Harrison et al., 1996). Therefore, it is highly unlikely that intact CP4 EPSPS protein would be present in loin tissue samples from pigs fed RR soybean meal. The present study demonstrated that an in vitro-digested preparation of CP4 EPSPS protein was detectable with a competitive ELISA configuration at concentrations similar to the intact protein (
4 ng/mL), whereas a more conventional sandwich ELISA demonstrated no reactivity with the partially digested CP4 EPSPS preparation up to 16 ng/mL. The CP4 EPSPS competitive immunoassay had an LOD of approximately 94 ng of CP4 EPSPS protein/g of pork loin tissue extract (94 ppb), and yet neither intact CP4 EPSPS nor immunoreactive fragments of the protein were detected in the loin homogenates from pigs fed RR soybean meal. It is important to note, however, that fully digested CP4 EPSPS would not be detected in this assay. Similar to the present results, it has been reported that the CP4 EPSPS protein is not detectable in a number of samples from laying hens fed RR soybean meal (Ash et al., 2000).
The present results showing the absence of detectable levels of fragments of either transgenic DNA or protein are consistent with the general understanding that these macromolecules undergo rapid and effective degradation in animal digestive tracts (Beever and Kemp, 2000). In addition, the safety of DNA and protein introduced into genetically enhanced agricultural products is based on strong scientific principles and premarket regulatory assessments (FAO/WHO, 1991; 1996; OECD, 1998; 2000). It is important to remember, nevertheless, that even if transgenic DNA is detected by a future technology, scientific evidence and opinion concludes that ingested transgenic DNA would not be different from ingestion of DNA already in foods, which is clearly safe (Jonas et al., 2001).
To summarize, previous studies and the present study show that fragments of the cp4 epsps transgene are not detectable in a variety of tissue samples from pigs, dairy cows, and broiler chickens fed a diet that includes RR soybeans or soybean meal.
|
Implications |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
|
Footnotes |
|---|
2 Roundup Ready soybean event 40-3-2.
3 We extend special thanks to E. Stanisiewski and G. Hartnell for arranging the collection of the animal tissue samples and reviewing the manuscript, and to R. Fuchs and M. Holland for their continuous support of this project and reviews of the manuscript.
Received for publication October 28, 2002. Accepted for publication February 12, 2003.
|
Literature Cited |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
This article has been cited by other articles:
|
|
 |
|
  A. L. Cerdeira and S. O. Duke The Current Status and Environmental Impacts of Glyphosate-Resistant Crops: A Review J. Environ. Qual., August 9, 2006; 35(5): 1633 - 1658. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Beagle, G. A. Apgar, K. L. Jones, K. E. Griswold, J. S. Radcliffe, X. Qiu, D. A. Lightfoot, and M. J. Iqbal The digestive fate of Escherichia coli glutamate dehydrogenase deoxyribonucleic acid from transgenic corn in diets fed to weanling pigs J Anim Sci, March 1, 2006; 84(3): 597 - 607. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. L. Cromwell, B. J. Henry, A. L. Scott, M. F. Gerngross, D. L. Dusek, and D. W. Fletcher Glufosinate herbicide-tolerant (LibertyLink) rice vs. conventional rice in diets for growing-finishing swine J Anim Sci, May 1, 2005; 83(5): 1068 - 1074. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. H. Phipps, E. R. Deaville, and B. C. Maddison Detection of Transgenic and Endogenous Plant DNA in Rumen Fluid, Duodenal Digesta, Milk, Blood, and Feces of Lactating Dairy Cows J Dairy Sci, December 1, 2003; 86(12): 4070 - 4078. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
—) or partially digested (—
—), was then analyzed in the competitive CP4 EPSPS ELISA (bottom).