Detection of a new non-MHC alloantigenic system (SLD) in pigs

A new, non-MHC cell membrane leucocyte alloantigen was detected in pigs by the complement dependent lymphocytotoxic technique. The new leucocyte system was designated SLD. Its product antigen SLD-1 was demonstrated to segregate independently of the SLA, SLB, SLC, A and E antigens. Family studies supplied evidence of a dominant inheritance of SLD-l. Since an allelic antigen could not be demonstrated, only two alleles for this locus are reported, namely SLD¹ and SLD^- . No evidence of linkage was detected between the above mentioned leucocyte alloantigenic systems and SLD. The antigen was detected on enriched suspensions of T and B cells from peripheral blood, but it was not detected on erythrocytes, granulocytes and thrombocytes.     

Polymorphism of the SLB serologically defined (non-MHC) alloantigenic system in pigs

Six new antigenic specificities (designated SLB-2 to SLB-7) detectable with polyclonal reagents in the complement-dependent lymphocytotoxic test were determined in the SLB alloantigenic system of pigs. The SLB specificities (including the previously described SLB-1) are controlled by 13 alleles. An analysis of a further 121 double backcross matings (with 817 offspring) confirmed that the products of SLB alleles segregated independently of the SLA haplotypes (swine MHC). In addition, in 112 families of the same mating type (with 746 progeny) the map distance of the SLB locus and the highly polymorphic L blood group locus was found to be 7.82 centimorgans in the heterogametic sex and 12.57 centimorgans in the homogametic sex.     

Detection of a new non-MHC alloantigenic system (SLD) in pigs

A new, non-MHC cell membrane leucocyte alloantigen was detected in pigs by the complement dependent lymphocytotoxic technique. The new leucocyte system was designated SLD. Its product antigen SLD-1 was demonstrated to segregate independently of the SLA, SLB, SLC, A and E antigens. Family studies supplied evidence of a dominant inheritance of SLD-1. Since an allelic antigen could not be demonstrated, only two alleles for this locus are reported, namely SLD1 and SLD-. No evidence of linkage was detected between the above mentioned leucocyte alloantigenic systems and SLD. The antigen was detected on enriched suspensions of T and B cells from peripheral blood, but it was not detected on erythrocytes, granulocytes and thrombocytes.     

Protein expression of lymphocytes in HLA-DR transgenic pigs by a proteomic approach

Matching donor and recipient human leucocyte antigen (HLA-II) could conquer cell-mediated rejection following transplantation. Transgenic pigs carrying HLA genes that "humanize" porcine organs, tissues, and cells were successfully generated. This study further clarifies the effect of HLA-DR transgenes on lymphocyte protein expression, via a proteomic approach. Lymphocytes were isolated from two HLA-DR transgenic pigs and three nontransgenic littermates on 157 d after birth. Soluble protein of 1x10(7) cells was separated using 2-DE. In total, 301 colloidal CBB-stained protein spots detected on all five 2-D gels were quantified. Thirty-three proteins were differentially expressed by a factor of 1.5. These proteins were subsequently identified by MALDI-TOF MS and MALDI-TOF/TOF MS/MS. These proteins were sorted into the following categories: chaperones, T-lymphocyte function, DNA/RNA processing, cytoskeleton-associated proteins, signal transduction, enzymes, and unknown. Previous studies have suggested that some of the identified proteins are associated with lymphocyte activation/proliferation. The identities of the unidentified spots and the systematic effect of these up- and down-regulated proteins on T-cell function in HLA-DR transgenic pigs require further exploration.     

Allotype polymorphism of serum globulins (Gp system) in pigs

By alloimmunization with whole serum reagents were obtained which revealed two new allotypes, GS and P30, in pigs. Evidence is presented that the allotypes G8, P30 and previously described G4 and G6 are controlled by three complex autoso-mal allelic genes forming a closed system. For this new allotype system we propose the designation Gp (globulin. pig). For alternative G4-GS and G6-P30, forming pairs of mutually exclusive alloantigens. it is proposed to use a standard designation GpA-Gpa and GpB-Gpb respectively.     

Map arrangement of the SLA chromosomal region and the J and C blood group loci in the pig

Linkage studies of three-point crosses (triple backcross matings) showed that the linear sequence of three of the pig's immunogenetic traits — the SLA major histocompatibility complex and the J and C blood group loci — is SLA-J-C . Andresen & Baker (1964) and Rasmusen (1965) described close linkage between the J and C blood group loci and respectively found their recombination frequency to be 5.29 ± 1.1 % and 7.00 ± 3.4 %; by combining the data the exact frequency was determined at 5.75 ± 0.79 % (Muir & Rasmusen, 1974). Later, linkage of the SLA major histocompatibility complex with both J (Hruban et al., 1976) and C (Hruban et al., 1977) erythrocytic loci was found. The maximum tabular lod score values were found in the recombination fraction Θ= 0.10 in comparison of SLA and J and in the fraction Θ= 0.20 in comparison of SLA and C (Hruban et al., 1977).     

An additional locus (PI4) in the protease inhibitor (PI) gene cluster of pigs

A further α-protease inhibitor system, PI4, was detected in porcine sera using either 2D agarose gel, pH 5.0-PAGE, pH 9.0, or ID PAGE followed by immunoblotting with rabbit anti-porcine PI2 or PI3 antisera. PI4 inhibited chymotrypsin, but not trypsin. Seven allelic variants of PI4 were described. By haplotyping of a-protease inhibitor systems in 52 complete families it was shown that PI4 locus belongs to the PI gene cluster. The probable order of the PI loci was: PI1, PO1A, PI2, PI4, PI3.     

A seroprevalence survey of Toxoplasma gondii in common wombats (Vombatus ursinus)

Serum was collected from 23 wild common wombats (Vombatus ursinus) on a pastoral property in the Southern Tablelands of New South Wales, Australia (231N 616E) between 3rd August 2001 and 25th March 2002. The serum was tested using three serological methods for antibodies to Toxoplasma gondii. Six animals (26.1%) were shown to have antibodies to T. gondii. The latex agglutination test proved to be less sensitive than the direct agglutination test or the modified agglutination test. This is the first serological survey of T. gondii in wombats. This is the first recorded use of the latex agglutination test on wombat serum. This study demonstrated the highest percentage of seropositive animals in any serological survey for T. gondii in marsupials.     

Two new common alleles of erythrocyte adenosine deaminase (ADA) in pigs

New adenosine deaminase variants ADA C and ADA D were found by means of agarose gel electrophoresis in pig erythrocytes. Family data supported the hypothesis that these are controlled by codominant alleles ADAC and ADAD. The ADAC allele was present in Large White (q = 0.076), Landrace (q = 0.037) and their crosses with other breeds. The ADAD allele was present in Duroc (q = 0.067) and its crosses. Allele frequencies for six pig breeds are given.     

New A system allele of red cell alloantigens in Paso Fino horses

Family data from Paso Fino horses support the existence of a new allele (Aabdf) in the A system of red cell alloantigens. Considering breeds throughout the world, the A system now consists of 13 alleles defined by reagents which serologically detect seven factors.     

中国两头乌猪品种内源性逆转录病毒基因研究

目的对5个中国两头乌猪品种(通城猪、东山猪、沙子岭猪、赣西两头乌猪和金华猪)及3个国外品种(大白猪、长白猪和杜洛克猪)猪内源性逆转录病毒(PERV)的核心蛋白(gag)基因、多聚酶(pol)基因、囊膜(env)基因的3个亚型A、B、C,分别从DNA和RNA水平上进行研究,以发现中国两头乌猪品种在异种器官移植中的资源优势。方法利用PCR方法在DNA水平上对PERV基因的三个亚型进行鉴定,并通过半定量PCR方法在RNA水平上检测通城猪和大白猪PERV各亚型在心、肝、脾、肺、肾、肌肉、脂肪、淋巴和脑组织中的表达谱。结果4个华中两头乌猪种中env-AB型为主要PERV亚型,分别占被测总数的92%～100%。在这4个品种中均没有检测到C亚型,金华猪以及3个国外猪种中均检测到了C亚型,病毒亚型种类也更丰富。半定量PCR实验结果显示gag、pol基因在两个品种9个组织中广泛表达,env-A在通城猪的心、肝、肺、脂肪和淋巴组织中表达量较低,env-B在通城猪的心脏和淋巴组织中表达量较低,而env-B在大白猪的肾脏中表达很低,其他所测8个组织中表达量都较高。结论通城猪、东山猪、赣西两头乌猪和沙子岭猪可以做为较佳的异种移植候选供体,具有良好的应用前景。     

Unusual D system inheritance in Anglo-Arab horse

An unusual D system phenogroup appeared in one family line of Anglo-Arab horse. This phenogroup probably originated from inheritance with an apparent absence of factors and was transmitted through successive generations.     

Efficient production of omega-3 fatty acid desaturase (sFat-1)-transgenic pigs by somatic cell nuclear transfer

Omega-3(ω-3) fatty acid desaturase transgenic pigs may improve carcass fatty acid composition. The use of transgenic pigs is also an excellent large animal model for studying the role of ω-3 fatty acids in the prevention and treatment of coronary heart disease and cancer. Transgenic pigs carrying synthesized fatty acid desaturase-1 gene (sFat-1) from Caenorhabditis briggsae by somatic cell nuclear transfer (SCNT) were produced for the first time in China. Porcine fetal fibroblast cells were transfected with a sFat-1 expression cassette by the liposome-mediated method. Transgenic embryos were reconstructed by nuclear transfer of positive cells into enucleated in vitro matured oocytes. A total of 1889 reconstructed embryos were transferred into 10 naturally cycling gilts. Nine early pregnancies were established, 7 of which went to term. Twenty-one piglets were born. The cloning efficiency was 1.1% (born piglets/transferred embryos). The integration of the sFat-1 gene was confirmed in 15 live cloned piglets by PCR and Southern blot except for 2 piglets. Expression of the sFat-1 gene in 12 of 13 piglets was detected with RT-PCR. The data demonstrates that an efficient system for sFat-1 transgenic cloned pigs was developed, which led to the successful production of piglets expressing the sFat-1 gene.     

Effects of deoxynivalenol (DON) on growth performance, nutrient digestibility and DON metabolism in pigs

Wheat infected naturally withFusarium, contaminated mainly with 18.53 mg DON per kg, was added to a total constant wheat proportion of 400g/kg diet. Control and DON contaminated feed was fed for 11 weeks underad libitum and restrictive feeding conditions to 48 pigs of both sexes, which were randomly divided into 4 groups. Effects on performance (live weight range between 26 and 100kg), duration of feed intake and blood parameters were investigated. Parallel to this study, a balance study was carried out to examine the effects on nutrient digestibility and DON metabolism. The group fed the DON contaminated rationad libitum consumed 15% less feed and gained 14% less live weight compared to thead libitum control group, while the feed to gain ratio was unaffected. Under restrictive feeding conditions (DON and control) pigs exhibited 33%, 25% and 10% lower feed consumption, live weight gain and feed to gain ratio, respectively, than the control group fedad libitum. Metabolizability of energy, digestibility of organic matter, crude protein, crude fat and N-retention were significantly increased by 4, 3, 6, 11 and 10%, respectively, in the DON group of the restrictively fed pigs. In average up to 43.2% of the ingested DON, as the parent toxin in both groups, was eliminated with the urine and up to 3.0% with faeces. DON fed animals needed more time to consume the restrictive ration than the control group. Presented at the 26^th Mykotoxin-Workshop in Herrsching, Germany, May 17–19, 2004. Financial support Deutsone Forschungsgemeingschaft     

曲古抑菌素A对克隆猪端粒长度的影响

端粒是染色体末端结构, 在细胞分裂时随着DNA复制而缩短, 体细胞核移植能不同程度地延长端粒长度, 但有些克隆动物端粒的长度在体细胞核移植过程中不能有效恢复, 因而这些克隆动物就会表现出早衰现象。文章发现克隆东北民猪以及eGFP、Mx和PGC1α转基因克隆猪的端粒长度与核供体成体成纤维细胞相比显著缩短(P<0.05), 表明体细胞核移植的重编程过程没能延长细胞的“寿命”。曲古抑菌素A(Trichostatin A, TSA)是一种去乙酰化酶抑制剂, 有研究表明其能提高某些物种的体细胞核重编程效率。为了使端粒长度有效恢复, 文章利用40 nmol/L TSA处理1细胞期猪克隆胚胎24 h, 结果发现, 与对照组相比, TSA处理能显著地提高克隆胚胎体外发育的囊胚率(16.35% vs. 2 7.09%, 21.60% vs. 34.90%, P<0.05), 而且囊胚期端粒长度也得到显著延长(P<0.05)。克隆胚胎移植受体后得到了TSA处理组与非处理组的克隆猪, 虽然TSA处理并没有提高克隆效率(1.3% vs. 1.7%, TSA vs. control), 但端粒长度与对照组和供体细胞相比均显著延长(P<0.05)。猪体细胞核移植不能有效恢复端粒长度, 但是TSA处理能有效延长克隆猪端粒长度。     

Mono-(2-ethylhexyl) phthalate (MEHP) induces testicular alterations in male guinea pigs at prepubertal stage

We have recently shown that MEHP induces spermatogenic cell apoptosis in guinea pigs at prepubertal stage in vitro. To evaluate the effects of MEHP on the testicular tissues of guinea pigs in vivo, we conducted this research work. Five weeks old male guinea pigs were used in this experiment. They received a single oral dose of 2000 mg/ml of MEHP in corn oil by gavage at a volume equal to 4 ml/kg. Control group received a similar volume of corn oil vehicle. Vehicle- and MEHP-treated guinea pigs were sacrificed at the interval of 3, 6, and 9 h, and the testicular tissues were processed for histopathological studies. Distinct histopathological changes were recognized in testes. Detachment and displacement of spermatogenic cells, thin seminiferous epithelia, vacuolization of Sertoli cells were prominent at 6 h after MEHP treatment. The lumina of the efferent ductules were frequently occupied with sloughed seminiferous epithelia from 6 to 9 h after MEHP treatment. Apoptotic spermatogenic cells appeared at 3 h in the control group. The incidence of apoptotic spermatogenic cells significantly increased (*p<0.05) from 3 to 9 h, and the maximal increase of apoptotic spermatogenic cells were observed at 9 h after MEHP treatment. Time-dependent increases of apoptotic spermatogenic cells was recognized throughout the experimental period. It may be suggested here that MEHP also induces spermatogenic cell apoptosis in guinea pigs in vivo and guinea pigs may be considered as a useful animal model for sensitivity test of the reproductive toxicity to some phthalate esters at their earlier stage in vivo.     

Molecular characterization of the human ABO blood group orthologus system in pigs

The selection and use of animals with blood group 0 in the process of transplanting pig organs or tissues into humans can positively contribute to the control of acute immune rejection due to differences in blood groups. Exon-specific PCRs for the porcine blood group A transferase gene against genomic DNA from either blood group A or 0 animals resulted in the amplification failure of the A0 blood group gene exon 8 from blood group 0 animals. To characterize the genetic abnormality in the genome of blood group 0 animals, we screened bacterial artificial chromosome (BAC) clones from a Korean native pig BAC library which had the blood group 0 allele, and carried out shotgun sequencing. The analysis showed that the 0 allele has a large deletion between exon 7 of the A0 blood group gene and the neighbouring SURF6. We also showed that the ABO blood group antigens in humans and the A0 blood group antigens in pigs are coded by mutations within the orthologous glycosyltransferase gene. In addition, we developed a multiplex genotyping method for the porcine A0 blood group gene.     

Production of transgenic miniature pigs by pronuclear microinjection

Miniature pig is an attractive animal for a wide range of research fields, such as medicine and pharmacology, because of its small size, the possibility of breeding it under minimum environmental controls and the physiology that is potentially similar to that of human. Although transgenic technology is useful for the analysis of gene function and for the development of model animals for various diseases, there have not yet been any reports on producing transgenic miniature pig. This study is the first successful report concerning the production of transgenic miniature pig by pronuclear microinjection. The huntingtin gene cloned from miniature pig, which is a homologue of candidate gene for Huntington's disease, connected with rat neuron-specific enolase promoter region, was injected into a pronucleus of fertilized eggs with micromanipulator. The eggs were transferred into the oviduct of recipient miniature pigs, whose estrus cycles were previously synchronized with a progesterone analogue. A total of 402 injected eggs from 171 donors were transferred to 23 synchronized recipients. Sixteen of them maintained pregnancy and delivered 65 young, and one resulted in abortion. Five of the 68 offspring (three of which were aborted) were determined to have transgene by PCR and Southern analysis. The overall rate of transgenic production was 1.24% (transgenic/injected eggs). This study provides the first success and useful information regarding production of transgenic miniature pig for biomedical research.