Mammals, including humans and mice, employ V(D)J recombination and somatic hypermutation (SHM) to diversify their immunoglobulin (Ig) variable region repertoires, while domestic poultry, such as chickens and ducks, rely heavily on gene conversion (GCV) to achieve this diversification. Given the differences in the arrangement of Ig gene loci between chickens/ducks and humans/mice, we posited that the structural arrangement of the Ig locus might play a pivotal role in driving the pronounced frequency of GCV in poultry. To test this hypothesis, we engineered a novel mouse model, Igλ Igκ DIgL, characterized by the incorporation of the entire duck IgL gene locus and the simultaneous absence of the mouse endogenous IgL locus. These transgenic mice produced chimeric antibodies comprising mouse heavy chains and duck light chains, thereby offering partial support for B cell development. However, when subjected to repeated immunization with a variety of antigens, the humoral immune response in Igλ Igκ DIgL mice was notably diminished compared to that in wild-type mice. High-throughput sequencing of IgL repertoires unveiled distinct differences between Igλ Igκ DIgL mice and ducks in terms of V gene utilization frequencies, characteristics of the CDR3 region (CDRL3), and nucleotide additions at junctions (NP nucleotides). Despite the increased expression of duck IgL due to the absence of endogenous mouse IgL, the level of GCV observed in duck IgL of Igλ Igκ DIgL mice (2.01%) remained significantly lower than that in ducks (35.3%). These findings collectively indicate that the structural configuration of the Ig locus may not be a critical determinant of GCV and provide valuable insights into the mechanisms underpinning the high frequency of GCV observed in poultry species.

In contrast to humans or rabbits, in which maternal IgG is transmitted to offspring prenatally via the placenta or the yolk sac, large domestic animals such as pigs, cows and sheep transmit IgG exclusively through colostrum feeding after delivery. The extremely high IgG content in colostrum is absorbed by newborns via the small intestine. Although it is widely accepted that the neonatal Fc receptor, FcRn, is the receptor mediating IgG transfer across both the placenta and small intestine, it remains unclear whether FcRn also mediates serum IgG transfer across the mammary barrier to colostrum/milk, especially in large domestic animals. In this study, using a FcRn knockout pig model generated with a CRISPR-Cas9-based approach, we clearly demonstrate that FcRn is not responsible for the IgG transfer from serum to colostrum in pigs, although like in other mammals, it is involved in IgG homeostasis and mediates IgG absorption in the small intestine of newborns.

Having a limited number of VH segments, cattle rely on uniquely long DH gene segments to generate CDRH3 length variation (3-70 aa) far greater than that in humans or mice. Bovine antibodies with ultralong CDRH3s (>50 aa) possess unusual structures and abilities to bind to special antigens. In this study, we replaced most murine endogenous DH segments with bovine DH genes, generating a mouse line termed B-DH. The use of bovine DH genes significantly increased the length variation of CDRH3 and consequently the Ig heavy chain repertoire in B-DH mice. However, no ultralong CDRH3 was observed in B-DH mice, suggesting that other factors, in addition to long DH genes, are also involved in the formation of ultralong CDRH3. The B-DH mice mounted a normal humoral immune response to various antigens, although the B-cell developmental paradigm was obviously altered compared with wild-type mice. Additionally, B-DH mice are not predisposed to the generation of autoantibodies despite the interspecies DH gene replacement. The B-DH mice reported in this study provide a unique model to answer basic questions regarding the synergistic evolution of DH and VH genes, VDJ recombination and BCR selection in B-cell development.

Despite great values in many applications, heavy chain-only antibodies (HcAbs) are naturally only produced in camelids and sharks, which are not easy to access and handle. Production of the type of antibodies in small laboratory animals would remarkably facilitate their applications. We previously reported a mouse line in which the CH1 exon of mouse γ1 was deleted that could express heavy chain-only IgG1 antibodies. However, these mice showed an extremely weak IgG1 response to specific antigens when immunized, and we could only achieve single VH domains with low affinity to antigens using these mice. One possibility is that the mouse germline VH repertoire was not sufficient to support the expression of functional heavy chain-only antibodies. In this study, we report the generation of a rat line in which the CH1 exon of the γ2a gene was removed and the γ1 and γ2b genes were silenced. Although the genetically modified rats expressed heavy chain-only IgG2a, they also exhibited a very weak IgG2a response to antigen immunization. Panning of a phage library constructed using IgG2a VH segments amplified from immunized rats identified antigen-specific single VH antibodies, which also exhibited much lower affinity than that of commercial mAbs. Together with our previous report, this study suggests that the simple genetic removal of the CH1 exon does not guarantee the successful expression of functional heavy chain-only antibodies.

Atypical TCR delta found in sharks, amphibians, birds, and monotremes and TCR mu found in monotremes and marsupials are TCR chains that use Ig or BCR-like variable domains (VH delta/V mu) rather than conventional TCR V domains. These unconventional TCR are consistent with a scenario in which TCR and BCR, although having diverged from each other more than 400 million years ago, continue to exchange variable gene segments in generating diversity for Ag recognition. However, the process underlying this exchange and leading to the evolution of these atypical TCR receptor genes remains elusive. In this study, we identified two TCR alpha/delta gene loci in the Chinese alligator (Alligator sinensis). In total, there were 144 V, 154 J alpha, nine J delta, eight D delta, two C alpha, and five C delta gene segments in the TCR alpha/delta loci of the Chinese alligator, representing the most complicated TCR alpha/delta gene system in both genomic structure and gene content in any tetrapod examined so far. A pool of 32 VH delta genes divided into 18 subfamilies was found to be scattered over the two loci. Phylogenetic analyses revealed that these VH delta genes could be related to bird VH delta genes, VH delta/V mu genes in platypus or opossum, or alligator VH genes. Based on these findings, a model explaining the evolutionary pattern of atypical TCR delta/TCR mu genes in tetrapods is proposed. This study sheds new light on the evolution of TCR and BCR genes, two of the most essential components of adaptive immunity.

AbstractAtypical TCRδ found in sharks, amphibians, birds, and monotremes and TCRμ found in monotremes and marsupials are TCR chains that use Ig or BCR-like variable domains (VHδ/Vμ) rather than conventional TCR V domains. These unconventional TCR are consistent with a scenario in which TCR and BCR, although having diverged from each other more than 400 million years ago, continue to exchange variable gene segments in generating diversity for Ag recognition. However, the process underlying this exchange and leading to the evolution of these atypical TCR receptor genes remains elusive. In this study, we identified two TCRα/δ gene loci in the Chinese alligator (Alligator sinensis). In total, there were 144 V, 154 Jα, nine Jδ, eight Dδ, two Cα, and five Cδ gene segments in the TCRα/δ loci of the Chinese alligator, representing the most complicated TCRα/δ gene system in both genomic structure and gene content in any tetrapod examined so far. A pool of 32 VHδ genes divided into 18 subfamilies was found to be scattered over the two loci. Phylogenetic analyses revealed that these VHδ genes could be related to bird VHδ genes, VHδ/Vμ genes in platypus or opossum, or alligator VH genes. Based on these findings, a model explaining the evolutionary pattern of atypical TCRδ/TCRμ genes in tetrapods is proposed. This study sheds new light on the evolution of TCR and BCR genes, two of the most essential components of adaptive immunity.

IgG subclass diversification is common in placental mammals. It has been well documented in humans and mice that different IgG subclasses, with diversified functions, synergistically regulate humoral immunity. However, our knowledge on the genomic and functional diversification of IgG subclasses in the pig, a mammalian species with high agricultural and biomedical importance, is incomplete. Using bacterial artificial chromosome sequencing and newly assembled genomes generated by the PacBio sequencing approach, we characterized and mapped the IgH C region gene locus in three indigenous Chinese breeds (Erhualian, Xiang, and Luchuan) and compared them to that of Duroc. Our data revealed that IGHG genes in Chinese pigs differ from the Duroc, whereas the IGHM, IGHD, IGHA, and IGHE genes were all single copy and highly conserved in the pig breeds examined. Most striking were differences in numbers of IGHG genes: there are seven genes in Erhualian pigs, six in the Duroc, but only five in Xiang pigs. Phylogenetic analysis suggested that all reported porcine IGHG genes could be classified into nine subclasses: IGHG1, IGHG2a, IGHG2b, IGHG2c, IGHG3, IGHG4, IGHG5a, IGHG5b, and IGHG5c. Using sequence information, we developed a mouse mAb specific for IgG3. This study offers a starting point to investigate the structure-function relationship of IgG subclasses in pigs.

In this study, using a dual-functional, piggyBac transposon-based system, we developed a method to systematically decipher the host genes that may be associated with porcine reproductive and respiratory syndrome virus (PRRSV) infection. A Marc145 cell library, which was randomly mutated by transfecting piggyBac plasmids, was challenged with PRRSV. The surviving cell clones were subjected to inverse PCR and high-throughput sequencing to map the integration sites of the transposon. Detailed annotation of the genes flanking the integration sites allowed us to generate a ranked list of candidate genes. Among the predicted genes with a high priority, four genes, CDK17, RNF168, BCL2L15, and TRIM33, were strongly correlated with PRRSV infection in both Marc145 cells and porcine primary alveolar macrophages. This study not only assists in identifying the genes essential for PRRSV infection but also confirms the possibility of using the piggyBac system to study other virus-host genetic interactions in a high-throughput manner.

The evolutionary origins of CD1d molecules and the T cell receptors (TCR) that recognize them currently remain unknown. CD1d molecules control the functions of a specialized subset of lymphocytes called semi-invariant natural killer T (type I NKT) cells. Type I NKT cells recognize both the self and non-self agonist —α-galactosylceramide (αGC) and related glycolipids— when presented by CD1d molecules. NKT cells express an invariant TCR α-chain (iTCRα) resulting from the mouse Vα14 or human Vα24 to Jα18 rearrangement. iTCRα pairs with the mouse Vβ8 (Vβ7, Vβ6, Vβ2) or the orthologous human Vβ11 TCR β-chain to create a functional NKTCR. Our phylogenetic analyses revealed that whilst the mouse Cd1 homologs have Mesozoic origin in they they evolved as far back asin the anole lizards, the orthologues of Cd1d and that of Vα14/Vα24 and Jα18 gene segments emerged with the decent of mammals. Moreover, the known avian CD1 orthologs lack the α3 domain and both the reptilian and the avian CD1 lack a functional endo/lysosomal-recycling motif critical for NKT cell development and function. As well, monotreme genomes lack Cd1d, Vα14/Vα24 and Jα18 orthologs and the marsupial Cd1d, Vα14/Vα24 and Jα18 orthologs harbor indels that would render the encoded proteins non-functional. Consistent with these findings, one of the most divergent Cd1d gene isolated from the nine-banded armadillo Dasypus novemcinctus and ectopically expressed in a cell line presented αGC and activated mouse NKT cell hybridomas. Hence, we conclude that the CD1d-restricted αGC-presentation in the immune system evolved more recently as an eutherian innovation.

The development of a rapid and efficient system to generate porcine monoclonal antibodies (mAbs) is an important step toward the discovery of critical neutralizing targets for designing rational vaccines against porcine viruses. In this study, we established a platform for producing porcine mAbs based on single cell technologies. First, we singled out an optimal donor from 507 pigs based on serum antibody neutralizing activity against porcine reproductive and respiratory syndrome virus (PRRSV). After identifying the contribution of IgG to the neutralizing activity, single CD45R+IgG+Ag+ B cells were sorted from peripheral blood mononuclear cells (PBMCs). Single B cell RT-PCR was performed using primers designed to cover the germline repertoire of the porcine VH/VL gene segments. Paired VH/VLs were cloned into a eukaryotic expression vector and transfected into 293T cells. We demonstrate that full-length porcine mAbs were produced, and antigen-specific mAbs were obtained after further validation. The approach reported in this study can be applied to generate porcine mAbs against any given antigen and may help with the screening of neutralizing antibodies against porcine pathogens.

In this study, we identified a pair of nonrearranging VJ-joined Ig superfamily genes, termed putative remnants of an Ag receptor precursor (PRARP) genes, in chicken. Both genes encode a single V-set Ig domain consisting of a canonical J-like segment and a potential immunoreceptor tyrosine-based inhibitory or switch motif in the cytoplasmic region. In vitro experiments showed that both genes were expressed at the cell surface as membrane proteins, and their recombinant products formed a monomer and a disulfide-linked homodimer or a heterodimer. These two genes were mainly expressed in B and T cells and were upregulated in response to stimulation with poly(I:C) in vitro and vaccination in vivo. Orthologs of PRARP have been identified in bony fish, amphibians, reptiles, and other birds, and a V-C1 structure similar to that of Ig or TCR chains was found in all these genes, with the exception of those in avian species, which appear to contain degenerated C1 domains or divergent Ig domains. Phylogenetic analyses suggested that the newly discovered genes do not belong to any known immune receptor family and appear to be a novel gene family. Further elucidation of the functions of PRARP and their origin might provide significant insights into the evolution of the immune system of jawed vertebrates.

Nano-antibodies possess great potential in many applications. However, they are naturally derived from heavy chain-only antibodies (HcAbs), which lack light chains and the CH1 domain, and are only found in camelids and sharks. In this study, we investigated whether the precise genetic removal of the CH1 exon of the γ1 gene enabled the production of a functional heavy chain-only IgG1 in mice. IgG1 heavy chain dimers lacking associated light chains were detected in the sera of the genetically modified mice. However, the genetic modification led to decreased expression of IgG1 but increased expression of other IgG subclasses. The genetically modified mice showed a weaker immune response to specific antigens compared with wild type mice. Using a phage-display approach, antigen-specific, single domain VH antibodies could be screened from the mice but exhibited much weaker antigen binding affinity than the conventional monoclonal antibodies. Although the strategy was only partially successful, this study confirms the feasibility of producing desirable nano-bodies with appropriate genetic modifications in mice.

The neonatal Fc receptor (FcRn) is involved in IgG metabolism and transport in placental mammals. However, whether FcRn is responsible for IgG transfer from maternal serum to colostrum/milk is controversial. Interestingly, large domestic animals, such as cows, pigs, sheep, and horses, in which passive IgG transfer is exclusively completed via colostrum/milk, all express an FcRn α-chain that is shorter in the cytoplasmic tail (CYT) than its counterparts in humans and rodents. To address whether the length variation has any functional significance, we performed in vitro experiments using the Transwell system with the MDCK cell line stably transfected with various FcRn constructs; these clearly suggested that truncation of the CYT tail caused a polar change in IgG transfer. However, we observed no evidence supporting functional changes in IgG in vivo using mice in which the FcRn CYT was precisely truncated. These data suggest that the length variation in FcRn is not functionally associated with passive IgG transfer routes in mammals.

All jawed vertebrates have four T cell receptor (TCR) chains that are expressed by thymus-derived lymphocytes and play a major role in animal immune defence. However, few studies have investigated the TCR chains of crocodilians compared with those of birds and mammals, despite their key evolutionary position linking amphibians, reptiles, birds and mammals. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization, evolution and expression of TRB and TRG loci in Alligator sinensis. According to the sequencing data, the Alligator sinensis TRB locus spans approximately 500 Kb of genomic DNA containing two D-J-C clusters and 43 V gene segments and is organized as V beta((39))-pJ beta 1-pC beta 1-pD beta 1-D beta 2- J beta 2((12))-C beta 2-V beta((4)), whereas the TRG locus spans 115 Kb of DNA genomic sequence consisting of 18 V gene segments, nine J gene segments and one C gene segment and is organized in a classical translocon pattern as V gamma((18)) -J gamma((9))-C gamma. Moreover, syntenic analysis of TRB and TRG chain loci suggested a high degree of conserved synteny in the genomic regions across mammals, birds and Alligator sinensis. By analysing the cloned TRB/TRG cDNA, we identified the usage pattern of V families in the expressed TRB and TRG. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed TRB and TRG sequences. Phylogenetic analysis revealed that TRB and TRG loci possess distinct evolutionary patterns. Most Alligator sinensis V subgroups have closely related orthologues in chicken and duck, and a small number of Alligator sinensis V subgroups have orthologues in mammals, which supports the hypothesis that crocodiles are the closest relatives of birds and mammals. Collectively, these data provide insights into TCR gene evolution in vertebrates and improve our understanding of the Alligator sinensis immune system. (C) 2018 Elsevier Ltd. All rights reserved.

近年来,受全球奶业市场形势影响,我国奶业市场持续动荡,进入2016年以来,全国生鲜乳价格保持低迷,国内外生鲜乳价格持续倒挂,乳制品进口量明显回升.北京市奶业市场作为都市型奶业的典型代表,发展形势基本与全国奶业市场持平,持续低迷的奶业市场促进北京市中小型奶牛养殖场加速转型升级,不断提高的乳制品质量和巨大的消费潜力推动了北京市奶业的稳定发展.

Previous studies on a limited number of birds suggested that the IgD-encoding gene was absent in birds. However, one of our recent studies showed that the gene was definitely expressed in the ostrich and emu. Interestingly, we also identified subclass diversification of IgM and IgY in these two birds. To better understand immunoglobulin genes in birds, in this study, we analyzed the immunoglobulin heavy chain genes in the zebra finch (Taeniopygia guttata) and Gentoo penguin (Pygoscelis papua), belonging respectively to the order Passeriformes, the most successful bird order in terms of species diversity and numbers, and Sphenisciformes, a relatively primitive avian order. Similar to the results obtained in chickens and ducks, only three genes encoding immunoglobulin heavy chain isotypes, IgM, IgA and IgY, were identified in both species. Besides, we detected a transcript encoding a short membrane-bound IgA lacking the last two CH exons in the Gentoo penguin. We did not find any evidence supporting the presence of IgD gene or subclass diversification of IgM/IgY in penguin or zebra finch. The obtained data in our study provide more insights into the immunoglobulin heavy chain genes in birds and may help to better understand the evolution of immunoglobulin genes in tetrapods.

Since 10,000 BC, continuous human selection has led to intense genetic and phenotypic changes in pig (Sus scrofa) domestication. Through whole genome analysis of 257 individuals, we demonstrated artificial unidirectional and bidirectional selection as the primary force to shape the convergent and divergent changes between Chinese domestic pigs (CHD) and European domestic pigs (EUD). We identified 31 genes in unidirectional selection regions that might be related to fundamental domestication requirements in pigs. And these genes belong predominantly to categories related to the nervous system, muscle development, and especially to metabolic diseases. In addition, 35 genes, representing different breeding preference, were found under bidirectional selection for the distinct leanness and reproduction traits between CHD and EUD. The convergent genetic changes, contributing physical and morphological adaption, represent the common concerns on pig domestication. And the divergent genetic changes reflect distinct breeding goals between Chinese and European pigs. Using ITPR3, AHR and NMU as examples, we explored and validated how the genetic variations contribute to the phenotype changes.

Recently, many immune-related genes have been extensively studied in ducks, but relatively little is known about their TCR genes. Here, we determined the germline and expressed repertoire of TCR genes in White Peking duck. The genomic organization of the duck TCRα/δ, TCRγ and unconventional TCRδ2 loci are highly conserved with their counterparts in mammals or chickens. By contrast, the duck TCRβ locus is organized in an unusual pattern, (Vβ) n -Dβ-(Jβ) 2 -Cβ1-(Jβ) 4 -Cβ2, which differs from the tandem-aligned clusters in mammals or the translocon organization in some teleosts. Excluding the first exon encoding the immunoglobulin domain, the subsequent exons of the two Cβ show significant diversity in nucleotide sequence and exon structure. Based on the nucleotide sequence identity, 49 Vα, 30 Vδ, 13 Vβ and 15 Vγ unique gene segments are classified into 3 Vα, 5 Vδ, 4 Vβ and 6 Vγ subgroups, respectively. Phylogenetic analyses revealed that most duck V subgroups, excluding Vβ1, Vγ5 and Vγ6, have closely related orthologues in chicken. The coding joints of all cDNA clones demonstrate conserved mechanisms that are used to increase junctional diversity. Collectively, these data provide insight into the evolution of TCRs in vertebrates and improve our understanding of the avian immune system.

SNPs detected by re-sequencing in the associated region of Rongchang pigs. Table S6. SNPs co-segregated with hearing loss phenotype in three MITFâ r/r pigs and three MITFâ R/R Rongchang pigs. Table S7. SNPs uniquely detected in Rongchang pigs, and homozygous in MITFâ r/r . (XLSX 465 kb)

Background: Genesis of novel gene regulatory modules is largely responsible for morphological and functional evolution. De novo generation of novel cis-regulatory elements (CREs) is much rarer than genomic events that alter existing CREs such as transposition, promoter switching or co-option. Only one case of de novo generation has been reported to date, in fish and without involvement of phenotype alteration. Yet, this event likely occurs in other animals and helps drive genetic/phenotypic variation.Results: Using a porcine model of spontaneous hearing loss not previously characterized we performed gene mapping and mutation screening to determine the genetic foundation of the phenotype. We identified a mutation in the non-regulatory region of the melanocyte-specific promoter of microphthalmia-associated transcription factor (MITF) gene that generated a novel silencer. The consequent elimination of expression of the MITF-M isoform led to early degeneration of the intermediate cells of the cochlear stria vascularis and profound hearing loss, as well as depigmentation, all of which resemble the typical phenotype of Waardenburg syndrome in humans. The mutation exclusively affected MITF-M and no other isoforms. The essential function of Mitf-m in hearing development was further validated using a knock-out mouse model.Conclusions: Elimination of the MITF-M isoform alone is sufficient to cause deafness and depigmentation. To our knowledge, this study provides the first evidence of a de novo CRE in mammals that produces a systemic functional effect.