Linkage haplotype for allotypic variants of porcine IgA and IgG subclass genes.

Six putative subclasses of expressed porcine IgG have been described from gene sequences and allotypic variants for five of these have been proposed. We tested this hypothesis by studying the transcription of these 11 variants in outbred hemizygous farm pigs. Since Cγ subclass genes are closely linked, they are most likely inherited as a haplotype. Since hemizygous pigs can only express genes encoded on one chromosome, identifying the expressed genes can indicate which allelic variants are linked as well as testing whether the putative alleles are indeed alleles or separate subclass genes. The procedure for producing B cell knockout pigs has recently been described; our study examines transcripts from the hemizygous parents and offspring generated by this technology. More than 570 Cγ gene clones from hemizygous animals were identified according to subclass and allotype by a combination of clone hybridization and sequencing. IgG3 accounted for 80% in newborn animals but <5% in adults. IgG1 accounted for ~50% of all clones recovered from adults and IgG4 was the least frequently recovered (4%). Results indicate that IgG1(b), IgG2(a), IgG3, IgG4(a), IgG5(a), and IgG6(a) are linked and also linked to IgA(a). This comprises a haplotype for domesticated swine. For simplicity, we propose that the current nomenclature for the allotypes of IgG1 be reversed so that all genes in the Cγ(a)-Cα(a) haplotype are designated "a".

Targeted disruption of the porcine immunoglobulin kappa light chain locus.

Inactivation of the endogenous pig immunoglobulin (Ig) loci, and replacement with their human counterparts, would produce animals that could alleviate both the supply and specificity issues of therapeutic human polyclonal antibodies (PAbs). Platform genetics are being developed in pigs that have all endogenous Ig loci inactivated and replaced by human counterparts, in order to address this unmet clinical need. This report describes the deletion of the porcine kappa (κ) light chain constant (Cκ) region in pig primary fetal fibroblasts (PPFFs) using gene targeting technology, and the generation of live animals from these cells via somatic cell nuclear transfer (SCNT) cloning. There are only two other targeted loci previously published in swine, and this is the first report of a targeted disruption of an Ig light chain locus in a livestock species. Pigs with one targeted Cκ allele (heterozygous knockout or ±) were bred together to generate Cκ homozygous knockout (-/-) animals. Peripheral blood mononuclear cells (PBMCs) and mesenteric lymph nodes (MLNs) from Cκ -/- pigs were devoid of κ-containing Igs. Furthermore, there was an increase in lambda (λ) light chain expression when compared to that of wild-type littermates (Cκ +/+). Targeted inactivation of the Ig heavy chain locus has also been achieved and work is underway to inactivate the pig lambda light chain locus.

Generation of antibody- and B cell-deficient pigs by targeted disruption of the J-region gene segment of the heavy chain locus.

A poly(A)-trap gene targeting strategy was used to disrupt the single functional heavy chain (HC) joining region (J(H)) of swine in primary fibroblasts. Genetically modified piglets were then generated via somatic cell nuclear transfer (SCNT) and bred to yield litters comprising J(H) wild-type littermate (+/+), J(H) heterozygous knockout (±) and J(H) homozygous knockout (-/-) piglets in the expected Mendelian ratio of 1:2:1. There are only two other targeted loci previously published in swine, and this is the first successful poly(A)-trap strategy ever published in a livestock species. In either blood or secondary lymphoid tissues, flow cytometry, RT-PCR and ELISA detected no circulating IgM(+) B cells, and no transcription or secretion of immunoglobulin (Ig) isotypes, respectively in J(H) -/- pigs. Histochemical and immunohistochemical (IHC) studies failed to detect lymph node (LN) follicles or CD79α(+) B cells, respectively in J(H) -/- pigs. T cell receptor (TCR)(ß) transcription and T cells were detected in J(H) -/- pigs. When reared conventionally, J(H) -/- pigs succumbed to bacterial infections after weaning. These antibody (Ab)- and B cell-deficient pigs have significant value as models for both veterinary and human research to discriminate cellular and humoral protective immunity to infectious agents. Thus, these pigs may aid in vaccine development for infectious agents such as the pandemic porcine reproductive and respiratory syndrome virus (PRRSV) and H1N1 swine flu. These pigs are also a first significant step towards generating a pig that expresses fully human, antigen-specific polyclonal Ab to target numerous incurable infectious diseases with high unmet clinical need.

The piglet as a model for B cell and immune system development.

The ability to identify factors responsible for disease in all species depends on the ability to separate those factors which are environmental from those that are intrinsic. This is particularly important for studies on the development of the adaptive immune response of neonates. Studies on laboratory rodents or primates have been ambiguous because neither the effect of environmental nor maternal factors on the newborn can be controlled in mammals that: (i) transmit potential maternal immunoregulatory factors in utero and (ii) are altricial and cannot be reared after birth without their mothers. Employing the newborn piglet model can address each of these concerns. However, it comes at the price of having first to characterize the immune system of swine and its development. This review focuses on the porcine B cell system, especially on the methods used for its characterization in fetal studies and neonatal piglets. Understanding these procedures is important in the interpretation of the data obtained. Studies on neonatal piglets have (a) provided valuable information on the development of the adaptive immune system, (b) lead to important advances in evolutionary biology, (c) aided our understanding of passive immunity and (d) provided opportunities to use swine to address specific issues in veterinary and biomedical research and immunotherapy. This review summarizes the history of the development of the piglet as a model for antibody repertoire development, thus providing a framework to guide future investigators.

Cloned pigs generated from cultured skin fibroblasts derived from a H-transferase transgenic boar.

Cloned pigs were produced from cultured skin fibroblasts derived from a H-transferase transgenic boar. One 90 day fetus and two healthy piglets resulted from nuclear transfer by fusion of cultured fibroblasts with enucleated oocytes. The cells used in these studies were subjected to an extensive culture time, freezing and thawing, and clonal expansion from single cells prior to nuclear transfer. PCR and FACS analysis determined that the cloned offspring contained and expressed the H-transferase transgene. Microsatellite analysis confirmed that the clones were genetically identical to the boar. The cell culture and nuclear transfer procedures described here will be useful for applications requiring multiple genetic manipulations in the same animal.

Lack of class I major histocompatibility antigens on trophoblast of periimplantation blastocysts and term placenta in the pig.

In this study, the pattern of expression of class I major histocompatibility (MHC) antigens and mRNA on periimplantation blastocysts and term placental tissue was determined for the pig. Class I MHC antigens could not be detected immunohistochemically either on extra-embryonic membranes or on the embryonic portion of Day 14, 16, 22, and 25 blastocysts. Nor could class I MHC antigens be detected on the outer trophoblast epithelium and inner endodermal surface of the chorioallantoic membrane or on the outer and inner surfaces of the amnion at term. However, MHC class I antigens were detected on the vascular mesoderm found in both the chorion and amnion at term, and in Day 25 extra-embryonic membranes. Uterine endometrial cells and tissues and maternal peripheral blood leukocytes stained strongly for class I MHC antigens. There was a large difference in the intensity of class I MHC mRNA signal, detected by Northern blot analysis, in embryo/fetus-derived tissues compared to that in maternal tissues. The embryos appeared to express even less class I MHC mRNA than did the extra-embryonic membranes. In addition, in situ hybridization of Day 16 blastocysts indicated class I MHC mRNA to be ubiquitously expressed at low levels in embryos and extra-embryonic tissues compared to uterine endometrial tissue controls. Taken together, these results indicate that class I MHC antigens are either not expressed on the surface of the extra-embryonic/fetal membranes during gestation in the pig or are expressed at very low levels, and that specific mRNA is expressed at correspondingly low levels.

Isolation and genetic characterization of the porcine apolipoprotein E gene.

The present report describes the isolation and genetic characterization of the porcine apolipoprotein E (apo-E) gene. A single positive recombinant phage clone containing a 10.7-kb insert was isolated from a porcine genomic library, and a 4.2-kb fragment was subcloned and sequenced. The 4.2-kb fragment contained the entire apo-E gene in addition to upstream and downstream sequences (GenBank accession no. 470240). The porcine apo-E gene is made up of four exons and three introns, and encodes a preapo-E protein comprised of a signal peptide of 18 amino acids and a mature protein of 299 amino acids. The porcine apo-E gene contains a (CG)13 microsatellite marker within intron three. This microsatellite is moderately polymorphic, and at least four alleles were evident at this locus among 10 animals from each of the Yorkshire, Hampshire, Landrace and Duroc breeds. Finally, localization of the porcine apo-E gene to chromosome 6 band q2.1 was determined by fluorescent in situ hybridization and confirmed by genetic linkage analysis.

Generation of transgenic porcine chimeras using primordial germ cell-derived colonies.

In mice, two pluripotent cell lines, embryonic stem (ES) cells and embryonic germ (EG) cells, have been identified. We present here results indicating that porcine EG cell lines can be isolated, genetically transformed, and utilized to make transgenic chimeras. Briefly, primordial germ cells (PGCs) were isolated from Day 25-27 fetuses and plated on STO feeder cells in Dulbecco's modified Eagle's medium:Ham's F-10 medium supplemented with 0.01 mM nonessential amino acids, 2 mM glutamine, 15% fetal bovine serum, 0.1 mM 2-mercaptoethanol, 40 ng/ml human stem cell factor, 20 ng/ml human basic fibroblast growth factor, and 20 ng/ml human leukemia inhibitory factor. For genetic transformation, cells were electroporated with a construct containing the green fluorescent protein under control of the cytomegalovirus promoter. After electroporation, cells were plated and later examined under fluorescein isothiocyanate excitation. Fluorescent colonies were selected for chimera generation. Blastocysts collected from gilts on Day 5 were injected with 10-15 transgenic PGC-derived cells and transferred into recipient gilts. Gilts were hysterectomized on Day 25, and fetal tissues were analyzed by Southern blotting. Three chimeras out of 20 fetuses analyzed were transgenic. Additionally, when one recipient gilt was allowed to go to term, one piglet with transgenic contribution was identified.

Treatment of gilts with leukocytes from the sire does not improve reproductive performance.

The effectiveness of pretreatment of gilts with leukocyte antigens on reproductive performance was studied. Two experiments were carried out that involved the treatment of gilts with leukocytes prior to artificial or natural insemination. Gilts were randomly assigned to one of three treatment groups. Group A gilts received two doses of 2 x 10(8) sire's leukocytes; one injected intra-peritoneal at first estrus and the other infused into the uterus immediately prior to insemination at the second estrus cycle. Group B gilts were similarly treated with the 'gilt's own' (autologous) leukocytes and Group C animals received phosphate buffered saline as controls. The same boars were used as sires across all three treatment groups. Gilts were slaughtered during the fifth week of pregnancy and their reproductive performance assessed. When data from the three groups were compared there were significant differences in embryo survival and placental weights between the sire's leukocyte treatment and PBS control groups. Embryo survival rate (No viable embryos/No corpora lutea x 100) at the fifth week of gestation was lower (p < 0.05) and placental weights were higher in the sire's leukocyte treatment group (p < 0.05). However, the latter may likely be due to fewer developing embryos. Embryo survival and placental weights did not vary significantly between the sire's and autologous leukocytes treatment groups. The treatment of gilts with leukocytes from the boars used as sires showed no improvement on subsequent reproductive outcome over that observed for the autologous leukocytes or PBS control treatments. Consequently, in a healthy well managed herd leukocytes treatment offers no advantage to pig reproductive function.

Isolation, characterization and chromosomal localization of the porcine ciliary neurotrophic factor (CNTF) gene.

In the mouse, ciliary neurotrophic factor (CNTF) maintains embryonic stem cells in an undifferentiated state; yet, the heterologous protein has no similar effects on porcine embryonic stem (ES) cells. Consequently, we cloned and sequenced the porcine CNTF gene and assigned it to chromosome 2. The CNTF gene was found to contain two exons, which encoded a deduced polypeptide of 200 amino acids in length with 83%, 82%, 82% and 81% amino acid similarity when compared to known sequences in the rabbit, rat, human and mouse, respectively. Eight non-conservative amino acid changes were identified in the porcine protein when compared with other species. Comparison of the 5' region of the porcine CNTF and other mammalian cytokines indicated the presence of several conserved transcription-factor binding motifs, suggesting their importance for controlling the specific expression of these proteins. In addition, CNTF was localized to porcine chromosomes by fluorescence in situ hybridization (FISH). Chromosome arm length ratios were calculated for 40 early metaphase chromosomes and 32 (80%) indicated that the pig CNTF gene is located on chromosome 2p1.6. This was confirmed by aligning R-banded FISH-labelled chromosomes to the standard porcine ideogram.

Advances in the generation of transgenic pigs via embryo-derived and primordial germ cell-derived cells.

The development of new technologies that would increase the efficiency for generation of transgenic livestock and would overcome some of the problems associated with random insertion of the transgene will greatly benefit animal agriculture. A potential alternative technology to pronuclear injection for the generation of transgenic pigs involves the isolation, culture and genetic manipulation of cell lines that can be reintroduced into the embryo for participation in the formation of the germ cells. We have isolated and cultured pig primordial germ cells (PGC) while maintaining them in an undifferentiated state as determined by morphology and alkaline phosphatase (AP) activity. More importantly, PGC-derived cells were stably transformed with the green fluorescent protein marker driven by the cytomegalovirus promoter. After visual identification of transgenic colonies, the pluripotential characteristics of the transgenic PGC-derived cells were tested by chimaera formation and to date we have identified, by genomic Southern blots, two chimaeric fetuses that contain tissues with the transgene incorporated into their chromosomes. To our knowledge, this is the first report of a chimaeric transgenic pig fetus obtained via a cultured cell line.

Partial purification and characterization of a protein in porcine follicular fluid which restricts sperm-egg interaction in vitro.

An attempt was made to isolate and characterize a component in preovulatory porcine follicular fluid (pFF) which has a restricting effect on sperm-egg interaction in vitro. Using the zona-free hamster ova (eggs) penetration assay as an in vitro test system, it was shown previously that the numbers of porcine spermatozoa attached to or penetrated into each egg and the number of eggs with sperm attached or penetrated decreased significantly as the concentration of pFF was increased in the culture medium. In the present study, the component in pFF having these effects was shown to be a heat stable, nonsteroidal substance which retained its activity after dialysis, lyophilization and gel filtration chromatography. The activity was also found to be present in preovulatory homologous serum. Separation of the material on protein type gel filtration columns with detection at 280 nm, together with the banding seen with Coomassie staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), suggests that it is a protein. Based on high pressure liquid chromatographic separation (HPLC) and SDS-PAGE analyses, the bioactivity could be due to a single protein of 87 kD or to one or more of three smaller proteins, possibly disaggregated products of the 87 kD protein, in the range of 26-28 kD.

A porcine trophoblast cell line that secretes growth factors which stimulate porcine macrophages.

We have previously described experiments in both the mouse and the human indicating that cytokines capable of activating macrophages (colony-stimulating factor-1 [CSF-1], granulocyte-macrophage colony-stimulating factor [GM-CSF], and interleukin-3 [IL-3]) are produced by, and/or stimulatory of, trophoblast cells in these species. In contrast to the complex hemochorial placenta of the mouse and humans, the pig has a simple diffuse type of placenta, designated as epitheliochorial. To determine whether similar phenomena might not apply to the porcine pregnancy, we have isolated a cell line, designated Jag-1, from the trophoblastic tips of Day 14 porcine embryos. We report here that this cell line is cytokeratin-positive, vimentin-negative, and therefore of epidermal origin. It also shares various morphological characteristics with porcine trophoblast as demonstrated at both the light and electron microscopic levels. In addition, Jag-1 cells and primary trophoblast tissue from Day 14 blastocyst do not express classical major histocompatibility (MHC) class I and class II antigens, a unique feature of trophoblast in many species. To determine the ability of this cell line to produce cytokines, we have developed an assay for porcine macrophage growth factors that utilizes uptake of tritiated thymidine. This assay responds positively to recombinant bovine GM-CSF and, more importantly, detects a similar activity in supernatants of the porcine trophoblast cell line and of Day 14 blastocysts. Thus porcine trophoblast cells, like their murine and human counterparts, produce and potentially interact with lymphohematopoietic cytokines that are traditionally associated with macrophages.

The effect of porcine follicular fluid on the interaction of boar spermatozoa with zona-free hamster ova.

Boar spermatozoa were cocultured with zona-free hamster ova (eggs) to assess the effects of preovulatory porcine follicular fluid (pFF) in the capacitation medium or gamete coculture (fertilization) medium (pFF; 0, 10 or 40% v/v) on subsequent sperm-egg interaction. Increasing pFF concentrations in the capacitation medium resulted in a progressive decrease in the average numbers of sperm attaching to or penetrating each ovum. When pFF was included in the fertilization medium, but not in the capacitation medium, the average numbers of sperm attaching to or penetrating each ovum and the percentage of ova with sperm attached decreased markedly with increasing pFF concentrations. The percentage of ova with greater than five sperm attached decreased from 84% to 13% and 0% with 0%, 10% and 40% pFF, respectively. Sperm attachment was completely inhibited in approximately 50% of the ova cocultured in 40% pFF. The percentage of ova penetrated by greater than five sperm decreased from 82% to 21% and 7% with 0%, 10% and 40% pFF, respectively. Preincubation of ova in 40% pFF prior to coculture with sperm also resulted in a reduction in sperm attachment and penetration. These results suggest that pFF contains substance(s) that alter the ability of boar spermatozoa to interact with the hamster ovum plasma membrane in vitro.