Insights into the function of n-3 PUFAs in fat-1 transgenic cattle
2017; Elsevier BV; Volume: 58; Issue: 8 Linguagem: Inglês
10.1194/jlr.m072983
ISSN1539-7262
AutoresXinfeng Liu, Zhuying Wei, Chunling Bai, Xiangbin Ding, Xin Li, Guanghua Su, Lei Cheng, Li Zhang, Hong Guo, Guangpeng Li,
Tópico(s)Animal Genetics and Reproduction
ResumoThe n-3 PUFAs have many beneficial effects on human health, including roles in immunity, neurodevelopment, and preventing cardiovascular disease. In this study, we established reliable model fat-1 transgenic cattle using transgenic technology and performed a systematic investigation to examine the function of n-3 PUFAs. Our results showed that expression of the fat-1 gene improved several biochemical parameters related to liver function and to plasma glucose and plasma lipid metabolism. Results of global gene and plasma protein expression analysis showed that 310 genes and 13 plasma proteins differed significantly in the blood of fat-1 transgenic cattle compared with WT cattle, reflecting their regulatory roles in the immune and cardiovascular systems. Finally, changes in the gut microflora were also noted in the fat-1 transgenic cattle, suggesting novel roles for n-3 PUFAs in the metabolism of glucose and lipids, as well as anti-stress properties. To the best of our knowledge, this is the first report using multiple parallel analyses to investigate the role of n-3 PUFAs using models such as fat-1 transgenic cattle. This study provides novel insights into the regulatory mechanism of fat-1 in the immune and cardiovascular systems, as well as its anti-stress role. The n-3 PUFAs have many beneficial effects on human health, including roles in immunity, neurodevelopment, and preventing cardiovascular disease. In this study, we established reliable model fat-1 transgenic cattle using transgenic technology and performed a systematic investigation to examine the function of n-3 PUFAs. Our results showed that expression of the fat-1 gene improved several biochemical parameters related to liver function and to plasma glucose and plasma lipid metabolism. Results of global gene and plasma protein expression analysis showed that 310 genes and 13 plasma proteins differed significantly in the blood of fat-1 transgenic cattle compared with WT cattle, reflecting their regulatory roles in the immune and cardiovascular systems. Finally, changes in the gut microflora were also noted in the fat-1 transgenic cattle, suggesting novel roles for n-3 PUFAs in the metabolism of glucose and lipids, as well as anti-stress properties. To the best of our knowledge, this is the first report using multiple parallel analyses to investigate the role of n-3 PUFAs using models such as fat-1 transgenic cattle. This study provides novel insights into the regulatory mechanism of fat-1 in the immune and cardiovascular systems, as well as its anti-stress role. The n-3 PUFAs are beneficial to human health. Previous studies have revealed that increased intake of n-3 PUFAs could reduce the risk of major human diseases, including cardiovascular disease, type 2 diabetes, and several types of cancer (1.Liu F. Li Z. Lv X. Ma J. Dietary n-3 polyunsaturated fatty acid intakes modify the effect of genetic variation in fatty acid desaturase 1 on coronary artery disease.PLoS One. 2015; 10: e0121255PubMed Google Scholar, 2.Psota T.L. Gebauer S.K. Kris-Etherton P. Dietary omega-3 fatty acid intake and cardiovascular risk.Am. J. Cardiol. 2006; 98: 3i-18iAbstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 3.White P.J. Arita M. Taguchi R. Kang J.X. Marette A. Transgenic restoration of long-chain n-3 fatty acids in insulin target tissues improves resolution capacity and alleviates obesity-linked inflammation and insulin resistance in high-fat-fed mice.Diabetes. 2010; 59: 3066-3073Crossref PubMed Scopus (144) Google Scholar, 4.Algamas-Dimantov A. Yehuda-Shnaidman E. Hertz R. Peri I. Bar-Tana J. Schwartz B. Prevention of diabetes-promoted colorectal cancer by (n-3) polyunsaturated fatty acids and (n-3) PUFA mimetic.Oncotarget. 2014; 5: 9851-9863Crossref PubMed Scopus (11) Google Scholar). Previous studies have also shown that n-3 PUFAs play a positive role in the immune system by mediating the improved inflammatory response (5.Harbige L.S. Fatty acids, the immune response, and autoimmunity: a question of n-6 essentiality and the balance between n-6 and n-3.Lipids. 2003; 38: 323-341Crossref PubMed Scopus (262) Google Scholar, 6.Gravaghi C. La Perle K.M. Ogrodwski P. Kang J.X. Quimby F. Lipkin M. Lamprecht S.A. Cox-2 expression, PGE(2) and cytokines production are inhibited by endogenously synthesized n-3 PUFAs in inflamed colon of fat-1 mice.J. Nutr. Biochem. 2011; 22: 360-365Crossref PubMed Scopus (61) Google Scholar, 7.Delpech J.C. Madore C. Joffre C. Aubert A. Kang J.X. Nadjar A. Layé S. Transgenic increase in n-3/n-6 fatty acid ratio protects against cognitive deficits induced by an immune challenge through decrease of neuroinflammation.Neuropsychopharmacology. 2015; 40: 525-536Crossref PubMed Scopus (70) Google Scholar). The n-3 PUFAs are a collection of PUFAs that include α-linolenic acid (ALA), DHA, and EPA. ALA is abundant in plant oils and can be obtained directly through the diet (8.Swanson D. Block R. Mousa S.A. Omega-3 fatty acids EPA and DHA: health benefits throughout life.Adv. Nutr. 2012; 3: 1-7Crossref PubMed Scopus (812) Google Scholar). Conversion of ALA in mammals can produce DHA and EPA through the desaturation-chain elongation pathway (9.Liu X. Pang D. Yuan T. Li Z. Li Z. Zhang M. Ren W. Ouyang H. Tang X. N-3 polyunsaturated fatty acids attenuates triglyceride and inflammatory factors level in hfat-1 transgenic pigs.Lipids Health Dis. 2016; 15: 89Crossref PubMed Scopus (12) Google Scholar). Although a greater conversion capacity for ALA to DHA was found in women than men, the synthesis efficiency is limited (10.Li Y. Tang Y. Wang S. Zhou J. Zhou J. Lu X. Bai X. Wang X.Y. Chen Z. Zuo D. Endogenous n-3 polyunsaturated fatty acids attenuate T cell-mediated hepatitis via autophagy activation.Front. Immunol. 2016; 7: 350Crossref PubMed Scopus (22) Google Scholar). Therefore, DHA and EPA are also primarily obtained from the diet. The fat-1 gene encodes n-3 PUFA desaturase, which can specifically convert n-6 PUFAs to n-3 PUFAs. Transgenic technology can be used to produce fat-1 transgenic domestic animals, which could then generate food to supply n-3 PUFAs for human consumption. In 2004, Kang et al. (11.Kang J.X. Wang J. Wu L. Kang Z.B. Transgenic mice: fat-1 mice convert n-6 to n-3 fatty acids.Nature. 2004; 427: 504Crossref PubMed Scopus (458) Google Scholar) reported the first fat-1 transgenic mice, which could synthesize n-3 PUFAs from n-6 PUFAs through constitutive expression of the fat-1 gene in vivo, showing that it would be feasible to obtain a rich supply of n-3 PUFAs from transgenic domestic animals. Subsequently, various fat-1 transgenic domestic animals have been generated using transgenic technology. These studies have focused more heavily on fat-1 transgenic pigs than either sheep or cattle. Lai et al. (12.Lai L. Kang J.X. Li R. Wang J. Witt W.T. Yong H.Y. Hao Y. Wax D.M. Murphy C.N. Rieke A. et al.Generation of cloned transgenic pigs rich in omega-3 fatty acids.Nat. Biotechnol. 2006; 24: 435-436Crossref PubMed Scopus (300) Google Scholar) generated the first fat-1 transgenic pigs in 2006 and demonstrated their elevated level of n-3 PUFAs, which were three times more abundant in the tail tissue of fat-1 transgenic pigs than in WT pigs. In 2010, Pan et al. (13.Pan D. Zhang L. Zhou Y. Feng C. Long C. Liu X. Wan R. Zhang J. Lin A. Dong E. et al.Efficient production of omega-3 fatty acid desaturase (sFat-1)-transgenic pigs by somatic cell nuclear transfer.Sci. China Life Sci. 2010; 53: 517-523Crossref PubMed Scopus (41) Google Scholar) generated 21 piglets by transgenic somatic cell nuclear transfer; 15 of these piglets survived, and 13 of these were confirmed to positively express the fat-1 gene. The first fat-1 transgenic cattle were produced in our laboratory in 2009. The levels of four types of n-3 PUFA (18:3 n-3, 20:5 n-3, 22:6 n-3, and 22:5 n-3) in the ear tissues of the fat-1 transgenic cattle were significantly higher than those in the same tissues of WT cattle, whereas three types of n-6 PUFAs (18:2 n-6, 20:4 n-6, and 22:5 n-6) were significantly lower (14.Wu X. Ouyang H. Duan B. Pang D. Zhang L. Yuan T. Xue L. Ni D. Cheng L. Dong S. et al.Production of cloned transgenic cow expressing omega-3 fatty acids.Transgenic Res. 2012; 21: 537-543Crossref PubMed Scopus (74) Google Scholar). Next, Zhang et al. (15.Zhang P. Liu P. Dou H. Chen L. Chen L. Lin L. Tan P. Vajta G. Gao J. Du Y. et al.Handmade cloned transgenic sheep rich in omega-3 fatty acids.PLoS One. 2013; 8: e55941Crossref PubMed Scopus (44) Google Scholar) generated three fat-1 transgenic sheep in 2013. High levels of n-3 PUFAs, as well as a low ratio of n-6/n-3 PUFAs, were observed in the heart, liver, spleen, lung, kidney, brain, ear, tail, and muscle tissues. These results indicated the fat-1 genes had a physiological function converting n-6 PUFAs into n-3 PUFAs. The mouse is a convenient model animal for experimental research. Therefore, fat-1 mice have been extensively employed to investigate the role of n-3 PUFAs in many diseases. Li et al. (10.Li Y. Tang Y. Wang S. Zhou J. Zhou J. Lu X. Bai X. Wang X.Y. Chen Z. Zuo D. Endogenous n-3 polyunsaturated fatty acids attenuate T cell-mediated hepatitis via autophagy activation.Front. Immunol. 2016; 7: 350Crossref PubMed Scopus (22) Google Scholar) used fat-1 mice rich in endogenous n-3 PUFAs to explore the protective effect of n-3 PUFAs in immune-mediated liver injury, showing that n-3 PUFAs limit concanavalin A-induced hepatitis via an autophagy-dependent mechanism. Additionally, an anti-tumor function of n-3 PUFAs in fat-1 mice was suggested by studies showing a reduction in colitis-associated colon cancer associated with a decreased inflammatory response (16.Jia Q. Lupton J.R. Smith R. Weeks B.R. Callaway E. Davidson L.A. Kim W. Fan Y.Y. Yang P.Y. Newman R.A. et al.Reduced colitis-associated colon cancer in fat-1 (n-3 fatty acid desaturase) transgenic mice.Cancer Res. 2008; 68: 3985-3991Crossref PubMed Scopus (118) Google Scholar). Recently, fat-1 mice have been used in several models of neurological disease, including Parkinson's disease (17.Bousquet M. Saint-Pierre M. Julien C. Salem N. Cicchetti F. Calon F. Beneficial effects of dietary omega-3 polyunsaturated fatty acid on toxin-induced neuronal degeneration in an animal model of Parkinson's disease.FASEB J. 2008; 22: 1213-1225Crossref PubMed Scopus (183) Google Scholar), Alzheimer's disease (18.Lebbadi M. Julien C. Phivilay A. Tremblay C. Emond V. Kang J.X. Calon F. Endogenous conversion of omega-6 into omega-3 fatty acids improves neuropathology in an animal model of Alzheimer's disease.J. Alzheimers Dis. 2011; 27: 853-869Crossref PubMed Scopus (63) Google Scholar), epilepsy (19.Taha A.Y. Huot P.S. Reza-López S. Prayitno N.R. Kang J.X. Burnham W.M. Ma D.W. Seizure resistance in fat-1 transgenic mice endogenously synthesizing high levels of omega-3 polyunsaturated fatty acids.J. Neurochem. 2008; 105: 380-388Crossref PubMed Scopus (35) Google Scholar), chronic inflammatory demyelinating disease of the central nervous system (20.Siegert E. Paul F. Rothe M. Weylandt K.H. The effect of omega-3 fatty acids on central nervous system remyelination in fat-1 mice.BMC Neurosci. 2017; 18: 19Crossref PubMed Scopus (33) Google Scholar), and stroke-related brain injury (21.Hu X. Zhang F. Leak R.K. Zhang W. Iwai M. Stetler R.A. Dai Y. Zhao A. Gao Y. Chen J. Transgenic overproduction of mega-3 polyunsaturated fatty acids provides neuroprotection and enhances endogenous neurogenesis after stroke.Curr. Mol. Med. 2013; 13: 1465-1473Crossref PubMed Scopus (28) Google Scholar). All fat-1 mice models were protected from neuronal damage when compared with their WT littermates (20.Siegert E. Paul F. Rothe M. Weylandt K.H. The effect of omega-3 fatty acids on central nervous system remyelination in fat-1 mice.BMC Neurosci. 2017; 18: 19Crossref PubMed Scopus (33) Google Scholar). In this study, we first confirmed the expression and target function of the fat-1 gene in transgenic cattle based on several parameters, including DNA, RNA, and protein and fatty acid properties. Furthermore, we used fat-1 cattle models to investigate the role of n-3 PUFAs using multiple methods, including measurement of blood biochemical parameters, levels of gene expression and plasma proteins in the blood; we also evaluated changes in the gut microflora. To the best of our knowledge, this is the first report using multiple parallel analyses to investigate the function of n-3 PUFAs using fat-1 transgenic cattle models. The present study provides a valuable reference as well as novel insights into the function of n-3 PUFAs. All procedures performed for this study were consistent with the National Research Council Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Inner Mongolia University. Using the previously constructed fat-1 gene expression vector, PST200, bovine fetal fibroblasts were transfected, positive cells were pooled to produce transgenic embryos using somatic cell nuclear transfer, and the highest quality transgenic blastocysts were then selected for embryo transfer; all procedures were performed using previously described methods (14.Wu X. Ouyang H. Duan B. Pang D. Zhang L. Yuan T. Xue L. Ni D. Cheng L. Dong S. et al.Production of cloned transgenic cow expressing omega-3 fatty acids.Transgenic Res. 2012; 21: 537-543Crossref PubMed Scopus (74) Google Scholar). Ear tissue samples were taken from transgenic calves at 2 months of age. Total DNA and RNA were extracted from these tissues following previously described methods (14.Wu X. Ouyang H. Duan B. Pang D. Zhang L. Yuan T. Xue L. Ni D. Cheng L. Dong S. et al.Production of cloned transgenic cow expressing omega-3 fatty acids.Transgenic Res. 2012; 21: 537-543Crossref PubMed Scopus (74) Google Scholar). The presence of the fat-1 gene in each DNA sample was confirmed using PCR. Expression of fat-1 mRNA was detected via RT-PCR. Primers specific for the fat-1 gene (5′-ATTGTCAGGGCGATGTAGGC-3′ and 5′-CGGCTATCTGGTGTGGAACA-3′) were used for PCR and RT-PCR. The amplification conditions for PCR and RT-PCR included 35 cycles of incubation at 94°C for 30 s, at 62°C for 30 s, and at 72°C for 40 s. The amplification products were subjected to electrophoresis on a 1.5% agarose gel. Subsequently, Western blotting was performed for detection of the fat-1-encoded protein based on a custom antibody (Genecreate, Wuhan, China). To obtain reliable data for this study, all cattle, including transgenic and WT cattle, were housed in a concrete-sided cowshed prior to sample collection, fed the same diet (commercial concentrated feed and wet corn silage), and monitored daily to ensure their health. Ear tissue samples were taken from six transgenic calves and six WT calves at 3 months of age to extract lipids, and PUFA extraction was performed using gas chromatography as previously described (14.Wu X. Ouyang H. Duan B. Pang D. Zhang L. Yuan T. Xue L. Ni D. Cheng L. Dong S. et al.Production of cloned transgenic cow expressing omega-3 fatty acids.Transgenic Res. 2012; 21: 537-543Crossref PubMed Scopus (74) Google Scholar, 22.Lu Y. Nie D. Witt W.T. Chen Q. Shen M. Xie H. Lai L. Dai Y. Zhang J. Expression of the fat-1 gene diminishes prostate cancer growth in vivo through enhancing apoptosis and inhibiting GSK-3 beta phosphorylation.Mol. Cancer Ther. 2008; 7: 3203-3211Crossref PubMed Scopus (41) Google Scholar). Blood was collected from the jugular vein of three surviving transgenic calves and three WT calves at 6 months of age; these calves had been given limited feed for 24 h prior to sample collection. Each blood sample was placed into a tube containing 5% ethylene-diamine-tetraacetic acid. Plasma was separated by centrifugation at 3,500 g/min and 4°C for 15 min. Using the same methods, plasma was collected from the three surviving transgenic cattle and three WT cattle at 18 months and 4 years of age. Next, blood biochemical indices, including those for liver function [aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH)], renal function [creatinine (CRE)], plasma glucose (GLU), and plasma lipids [triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), and low density lipoprotein cholesterol (LDL-C)] were measured using the fully automatic biochemical analyzer, Glamour 3000 (Misiones Bernal, Buenos Aires, Argentina). Total RNA was extracted from the blood of three transgenic cattle and three WT cattle using the Trizol extraction protocol and purified using an RNeasy mini kit (Qiagen, Germany), following the manufacturer's protocol. For quality control, total RNA was quantified using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific), and RNA integrity was assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies). After completion of quality control procedures, total RNA was reverse transcribed into double-stranded cDNA; next, cRNA was synthesized, labeled with cyanine-3-CTP, purified, and hybridized to the bovine gene expression microarray (4 × 44K, Design ID: 023647). After elution onto hybridized arrays, the arrays were scanned at 5 μm using the Agilent scanner G2505C (Agilent Technologies). Feature Extraction software (version 10.7.1.1, Agilent Technologies) was used to analyze the array images and obtain raw data, whereas Gene Spring software was employed to perform general analysis. The raw data were normalized using the quantile algorithm. Gene expression data were deposited into the NCBI Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo) and can be accessed via accession number GSE66651. Differentially expressed genes were then identified based on fold changes. P values were calculated using the t-test, and the false discovery rate (FDR) was calculated to correct the P values using the R statistical package. The threshold for differentially expressed genes was set as a fold change >2.0, P < 0.01, and FDR value <0.05. Hierarchical clustering was performed to visualize gene expression patterns among the samples. GO analysis was used to determine the roles of these differentially expressed mRNAs, and biological functions with a P < 0.05 were considered statistically significant. Blood was collected in heparinized tubes from three transgenic cattle and three WT cattle. All six cattle were allowed limited feed for 24 h prior to sample collection. Whole blood was centrifuged at 7,000 g/min and 4°C for 10 min to obtain plasma, which was stored at −80°C until two-dimensional gel electrophoresis (2-DE) was performed. Equal amounts of plasma from three WT cattle were pooled to obtain the control group sample. The total plasma protein concentration was measured via the Bradford method using a protein assay reagent (Bio-Rad, Hercules, CA). The 2-DE was carried out based on the methods used in previous reports (23.Ding J. Berryman D.E. Kopchick J.J. Plasma proteomic profiles of bovine growth hormone transgenic mice as they age.Transgenic Res. 2011; 20: 1305-1320Crossref PubMed Scopus (20) Google Scholar). After 2-DE was performed, differentially expressed protein spots were screened based on the threshold of >1.5-fold change and a P value <0.05. These differentially expressed protein spots were identified using mass spectrometry, as previously described (23.Ding J. Berryman D.E. Kopchick J.J. Plasma proteomic profiles of bovine growth hormone transgenic mice as they age.Transgenic Res. 2011; 20: 1305-1320Crossref PubMed Scopus (20) Google Scholar). The annotation of the identified differential proteins was performed using the online tool, STRING (http://string-db.org/). Interactions among these differentially expressed proteins were also predicted using this tool. Common interaction proteins identified from the three transgenic cattle were extracted. These common interaction proteins were subjected to GO and KEGG analyses to gain insight into the effects on the three fat-1 transgenic cattle at the plasma protein level. Fecal samples were obtained from three transgenic cattle and three WT cattle by rectal palpation using sterile technique on the same morning. During collection, the outside air temperature was 15–18°C, and each sample was transferred into a separate sterilized container and immediately stored at 4°C, followed by long-term storage at −80°C until DNA was extracted. None of the cattle had received antibiotics within the past 3 months, and none had experienced gastrointestinal or acute disease. All cattle were housed in a concrete-sided cattle shed for 1 month prior to sample collection, were fed the same diet (commercial concentrate feed and wet corn silage), and were monitored every day to ensure their health. Total genomic DNA was extracted from fecal samples using a QIAamp stool DNA mini kit, in accordance with the manufacturer's protocol (Qiagen; 51504). Pyrobest DNA polymerase (Ta-KaRa, DR500A) was used for amplification of the V4 hypervariable region of the 16S rRNA gene from microbial genomic DNA. Next, the V4 amplicons were sequenced using the paired-end method on an Illumina MiSeq sequencer with a six-cycle index read. The raw sequence data were deposited into the NCBI database (https://www.ncbi.nlm.nih.gov/home/submit.shtml) and can be accessed via accession number SUB2038674. Sequence reads were trimmed so that the average Phred quality score for each read was greater than 30 and the read was longer than 50 bp; after trimming, these reads were assembled using Flash software (http://ccb.jhu.edu/software/FLASH/), and reads that could not be assembled were discarded. Only reads with series of consecutive identical bases shorter than 6 bp and without ambiguous bases were used for further analysis. Sequence clustering was performed using UCLUST (QIIME) with a similarity cutoff of 99% to form operational taxonomic units (OTUs). Next, the number and abundance of OTUs were determined for all samples. The species diversity (Chao1, ACE, Simpson, Shannon, and Coverage) of gut microbes was analyzed using the single summary command in MOTHUR software (http://www.mothur.org/). Differences in species and their distribution in the gut microbiota were analyzed based on the abundance profiles in transgenic and WT cattle. All data are expressed as mean values ± SD. The results were analyzed using Student's t-test, and differences were considered significant at either P < 0.05 or P < 0.01. In total, 1,156 reconstructed oocytes were produced after transferring fat-1 transgenic cells into enucleated oocyte cytoplasts. The cleavage and blastocyst rates were 80% and 32%, respectively. In total, 157 blastocysts were transferred to 106 synchronized recipient cattle. Pregnancy rates were 37.7% (40/106), 28.3% (30/106), and 18.8% (20/106) at 60, 90, and 210 days, respectively. In total, nine female calves were delivered naturally 280–286 days after transfer (Table 1). Of these nine calves, six fat-1 transgenic cattle were identified based on DNA, RNA, and protein analyses, and they were designated FD001, FD002, FD003, FD004, FD005, and FD006, respectively (Fig. 1A–C). At the present time, three of the fat-1 transgenic cattle (FD002, FD005, and FD006) are still alive and healthy. The survival rate of transgenic cattle was 33.3%.TABLE 1Development of bovine transgenic cloned embryos in vitroItemTransferred Blastocysts (n)Recipient Cattle (n)60-day Pregnancy (% Recipients)120-Day Pregnancy (% Recipients)210-Day Pregnancy (% Recipients)Number Birth (% Recipients)Number Survived (% Births)Total15710640 (37.7)30 (28.3)20 (18.8)9 (8.5)3 (33.3) Open table in a new tab To assess the activity of the fat-1 gene in transgenic cattle, fatty acids in the ear tissues were analyzed. As shown in Fig. 1D, various changes were found in the 14 types of fatty acid. Among the saturated fatty acids, eicosanoic acid, tricosanoic acid, and tetracosanoic acid increased significantly compared with the levels in the WT cattle used as the control group. The concentrations of two n-3 PUFAs, 5,8,11,14,17-EPA and 4,7,10,13,16,19-DHA were elevated in transgenic cattle compared with WT cattle, and the difference between the two groups for DHA was significant. On the other hand, the concentration of 9,12,15-octadecatrienoic acid (ALA) was lower in transgenic cattle than in WT cattle, but this difference was not significant. Levels of the n-6 PUFAs, 9,12-octadecenoic acid and 5,8,11,14-eicosatetraenoic acid, were significantly reduced in transgenic cattle compared with WT cattle, whereas both γ-linolenic acid and 15-tetracosenoic acid were increased significantly. Total n-6 PUFAs were significantly reduced and total n-3 PUFAs were significantly elevated in the transgenic cattle. However, the degree of reduction of n-6 PUFAs was greater than the degree of increase of n-3 PUFAs in the transgenic cattle. The n-6/n-3 PUFA ratio, which is approximately 1:1, was significantly reduced in the transgenic cattle. These results indicated that the expected function of the delivered fat-1 gene was successfully established using transgenic cattle. The results of blood biochemical levels for both fat-1 transgenic and WT cattle are shown in Table 2, which shows that only ALT, the biochemical parameter representing liver function, was significantly lower in transgenic cattle at 6 months than in WT cattle of the same age. When these cattle reached 18 months of age, AST, GLU, TC, and LDL-L were significantly reduced in the transgenic cattle relative to the WT cattle. Only GLU was significantly lower in the transgenic cattle than in the WT cattle when the cattle were 4 years old.TABLE 2Comparison of the blood biochemical parameters between the fat-1 transgenic cattle and WT cattleCalf (6 months old)Adult Cattle (18 months old)Adult Cattle (4 years old)ItemFDWTFDWTFDWTAST (U/l)63.34 ± 23.5169.20 ± 2.6177.98 ± 13.45aP < 0.05, WT cattle compared with the transgenic cattle.91.02 ± 3.7283.35 ± 9.8492.01 ± 2.74ALT (U/l)13.90 ± 1.91aP < 0.05, WT cattle compared with the transgenic cattle.16.63 ± 0.7535.90 ± 1.3538.27 ± 0.3536.08 ± 0.3837.46 ± 2.59LDH (U/l)693.33 ± 105.08785.33 ± 70.00871.7 ± 79.31993.37 ± 133.55894.16 ± 82.17969.41 ± 102.49CRE (μmol/l)55.33 ± 31.8248.67 ± 9.6189.67 ± 15.5766.67 ± 13.2088.56 ± 13.6968.11 ± 10.84GLU (mmol/l)4.73 ± 0.254.80 ± 0.953.53 ± 0.12aP < 0.05, WT cattle compared with the transgenic cattle.4.13 ± 0.213.63 ± 0.24aP < 0.05, WT cattle compared with the transgenic cattle.4.16 ± 0.16TG (mmol/l)0.10 ± 0.000.17 ± 0.060.23 ± 0.120.23 ± 0.060.22 ± 0.050.27 ± 0.03TC (mmol/l)2.10 ± 0.362.30 ± 1.232.03 ± 0.06aP < 0.05, WT cattle compared with the transgenic cattle.2.90 ± 0.052.14 ± 0.052.66 ± 0.48HDL-C (mmol/l)1.82 ± 0.121.88 ± 1.031.85 ± 0.072.45 ± 0.482.00 ± 0.102.41 ± 0.34LDL-C (mmol/l)0.14 ± 0.090.17 ± 0.050.19 ± 0.03aP < 0.05, WT cattle compared with the transgenic cattle.0.31 ± 0.080.24 ± 0.030.29 ± 0.05Plasma content of AST, ALT, LDH, CRE, GLU, TG, TC, HDL-C, and LDL-C in the WT cattle and transgenic cattle.a P < 0.05, WT cattle compared with the transgenic cattle. Open table in a new tab Plasma content of AST, ALT, LDH, CRE, GLU, TG, TC, HDL-C, and LDL-C in the WT cattle and transgenic cattle. In the present study, only the FD006 fat-1 transgenic animal was used for identification of the integration site and copy number using high-throughput sequencing (see supplemental Materials and Methods). Through a BLAST search of these sequencing reads, we obtained four bridging paired-end reads in which one end mapped to chromosome 16 of the bovine genome and the other end mapped to the fat-1 vector region. The four bridging paired-end reads were split for further analysis of the specific integration break points. The right boundary was located between position 15726078 of chromosome 16 and position 6475 of the inserted fat-1 vector (supplemental Fig. S1). However, we did not obtain bridging paired-end reads for the left boundary between chromosome 16 and the fat-1 vector region. More interestingly, introduction of a 10-nucleotide portion of the bovine genome at insertion site 15726078 of chromosome 16 was also observed, which is a characteristic signature of transgene integration (supplemental Fig. S1). To verify the transgene integration site, event-specific PCR was performed on FD006 DNA samples (supplemental Fig. S2). The sequencing depths for insertion site 15726078 of chromosome 16 and insertion site 6475 of fat-1 vector were 6× and 11×, respectively (supplemental Fig. S3). Therefore, we speculate that the transgene is a single copy. This result represents preliminary work to identify the transgene integration site and copy number in FD006. Identification of the integration site and copy number for two other transgenic cattle (FD002 and FD005) and analysis of genetic stability in these cattle will be performed in subsequent studies. Next, the gene expression patterns of the fat-1 transgenic and WT cattle were compared using an expression profile microarray. In total, 43,711 transcript sequences were used as probes. The results showed that 310 transcripts differed significantly in expression (P < 0.01, FDR < 0.05), reflecting up to a 2-fold change between the fat-1 transgenic and WT cattle (Fig. 2A). Of these 310 transcripts, 124 were significantly upregulated in fat-1 transgenic cattle compared with WT cattle (Fig. 2A). After hierarchical clustering analysis of the 310 genes, all three transgenic cattle showed consistent genetic backgrounds, which differed from the expression pattern of the WT cattle (Fig. 2B). To gain further insight into the potential influence of these 310 differentially expressed genes, GO enrichment analysis was performed. Twenty-four GO terms were significantly enriched, including eight GO terms representing biological process
Referência(s)