Ki Duk Song

A recombinant Eimeria protein inducing interferon-γ production: comparison of different gene expression systems and immunization strategies for vaccination against coccidiosis.

A rabbit antiserum against an 18- to 27-kD native protein fraction (F3) from Eimeria acervulina merozoites identified a cDNA (3-1E) containing a 1086-base pair insertion with an open reading frame of 170 amino acids (predicted molecular weight, 18,523). The recombinant 3-1E cDNA expressed in Escherichia coli produced a 60-kD fusion protein and a 23-kD protein after factor Xa treatment of the fusion protein. Both proteins were reactive with the F3 antiserum by western blot analysis. A rabbit antiserum against a synthetic peptide deduced from the amino acid sequence of the 3-1E cDNA reacted with a 27-kD recombinant 3-1E protein expressed in Sf9 insect cells and a 20-kD native protein expressed by E. acervulina sporozoites and Eimeria tenella sporozoites and merozoites. By immunofluorescence staining, a monoclonal antibody produced against the recombinant 3-1E protein reacted with sporozoites and merozoites of E. acervulina, E. tenella, and Eimeria maxima. Spleen lymphocytes from E. acervulina-immune chickens showed antigen-specific proliferation and interferon (IFN)-gamma production upon stimulation with the recombinant 3-1E protein, indicating that the protein activates cell-mediated immunity during coccidiosis. Immunization of chickens with either the E. coli- or Sf9-expressed recombinant 3-1E protein with adjuvant, or direct injection of the 3-1E cDNA, induced protective immunity against live E. acervulina. Simultaneous injection of the recombinant 3-1E protein, or the 3-1E cDNA, with cDNAs encoding chicken IFN-gamma or interleukin (IL)-2/15 further enhanced protective immunity. These results indicate that the recombinant E. acervulina 3-1E cDNA or its polypeptide product may prove useful as vaccines against avian coccidiosis.

Molecular and functional characterization of chickenIL-15

Abstract A cDNA encoding chicken interleukin-15 was cloned from a CD4 + T cellhybridoma expression library by screening with a rabbit antibody against a protein fraction ofconditioned medium containing T cell growth promoting activity. The chicken IL-15 cDNAcontains an open reading frame of 143 amino acids with a single potential N-linked glycosylationsite. The predicted m.w. of the encoded protein [16] matched the size of animmunoreactive band on Western blots of E. coli expressing the recombinant IL-15.Amino acid and nucleotide sequence analyses of chicken IL-15 revealed 31% and 46% identitywith bovine IL-15 respectively and lesser homologies to other mammalian IL-15s. Chicken IL-15contained all 4 highly conserved cysteine residues present in mammalian IL-15 sequences.RT-PCR demonstrated that the chicken IL-15 gene is expressed in many tissues including spleen,intestine, and muscle and in established macrophage, T lymphoma and fibroblast cell lines.Activation of spleen cells with Con A enhanced the expression of IL-15 gene transcripts in atime-dependent manner. CHO-K1 cells transfected with the chicken IL-15 cDNA secreted abiologically active protein supporting the growth of Con A activated spleen lymphocytes.Continuous culture of spleen Con A lymphoblasts with chicken IL-15 over two months resulted inan enriched T lymphocyte population expressing the gdTCR, CD8a, and CD3 cell surfaceantigens.

How many markers are enough? Factors influencing parentage testing in different livestock populations.

Reliability of parentage test panels is usually based on its power to exclude wrong parentage assignments based on allele frequencies. We evaluated the rates of false exclusions and inclusions in parentage assignments, and how these results are affected by allele frequencies, panel sizes and the number of allowed mismatches. We also evaluated the reliability of parentage testing by comparing populations with distinct genetic backgrounds using pure and composite families of cattle and sheep. Allowing for 1% genotype mismatches in true parent-offspring relations provided the best compromise between false-positive and false-negative assignments. Pure breeds needed at least 200-210 single-nucleotide polymorphism (SNP) markers to correctly assign relations, but between 700 and 890 markers to avoid assigning incorrect relationships. Composite breeds needed between 220 (sheep) and 500 (cattle) markers for correct assignment; 680 (cattle) to 4400 (sheep) SNPs were needed to eliminate false-positive assignments. Allowing 0% genotype mismatches decreased false-positive but increased false-negative assignments, whilst a higher threshold of 2% showed the opposite effects. Panels with high minor allele frequencies (0.35-0.45) provided the best chance for correct parentage resolutions requiring fewer markers. Further, we propose that a dynamic threshold would allow adapting to population specific error rates. A comparison to the performance of the official International Society for Animal Genetics SNP panel for cattle and a recently published SNP panel for sheep showed that randomly selected markers performed only slightly worse for the applied parentage test based on opposing homozygotes. This suggests that even with carefully selected panels, only marginal assignment improvements are obtainable for a particular number of SNPs. The main point for improvement is the number of markers used. We recommend using at least 200 SNP markers for parentage testing if the aim is to reduce false-negative results. To fully exclude false positives at least 700 markers are required.

Development of a type II diabetic mellitus animal model using Micropig

Diabetes, which has shown an explosive increase in terms of its incidence, is regarded as a serious disease that must be overcome for the sake of human life. Among animal models used for testing of drug efficacy, the mini-pig model has shown a rapid upload due to its many similarities with human, particularly concerning the pharmacokinetics of compounds after subcutaneous administration, the structure and function of the gastrointestinal tract, the morphology of the pancreas, and overall metabolic status. Based on these various advantages, we sought to develop an animal model of type II diabetic mellitus using the Micro-pig, which differs from other miniature pigs. We used six male Micro-pigs for induction of a moderate insulin deficient model with nicotinamide (NIA)/streptozotocin (STZ) treatment and three animals for control. For evaluation of incidence of type II diabetes, we measured blood glucose level, and performed oral glucose tolerance test and immunohistochemistry on pancreatic tissue using insulin antibody. Compared to control animals, all animals treated with NIA/STZ showed high levels of glucose and low levels of insulin. In addition, we observed the partially destroyed beta cell population from tissue of the pancreas in treated animals. Based on these results, we report that the Micro-pig model developed in this study can be used for testing of the efficacy of therapeutic agents for treatment of Type 2 diabetic mellitus.

Generation and characterization of recombinant ScFv antibodies detecting Eimeria acervulina surface antigens.

In our previous attempt to generate monoclonal antibodies (MAbs) against coccidia parasites that more accurately reflect the natural avian humoral immune response, we produced two chicken B-cell hybridomas, 5D11 and 2-1. While both cell lines secreted antibodies reactive with sporozoites of Eimeria acervulina, they were produced in yields too low to conduct meaningful in vivo studies. To circumvent this problem, we produced four single chain variable fragment (scFv) antibodies from the V(H) and V(L) genes of hybridomas 5D11 and 2-1. The concentration of these recombinant antibodies expressed in E. coli and purified to homogeneity was 5-6 mg/L. Three of the antibodies exhibited antigen binding specificity to Eimeria surface antigens equivalent to that of the native MAbs. Nucleotide sequence analysis of the V(L) genes from hybridomas 5D11 and 2-1 and genomic DNA revealed vestiges of gene conversion with V(lambda) pseudogenes. These recombinant scFv antibodies will prove useful for further characterization of natural Eimeria surface antigens as potential vaccine candidates.

Fibrates inhibit the apoptosis of Batten disease lymphoblast cells via autophagy recovery and regulation of mitochondrial membrane potential

Batten disease (BD; also known as juvenile neuronal ceroid lipofuscinosis) is a genetic disorder inherited as an autosomal recessive trait and is characterized by blindness, seizures, cognitive decline, and early death resulting from the inherited mutation of the CLN3 gene. Mitochondrial oxidative stress, endoplasmic reticulum (ER) stress, disrupted autophagy, and enhanced apoptosis have been suggested to play a role in BD pathogenesis. Fibrates, a class of lipid-lowering drugs that induce peroxisome proliferator-activated receptor-α (PPAR-α) activation, are the most commonly used PPAR agonists. Assuming that fibrates have a neuroprotective effect, we studied the effects of fibrates, fenofibrate, bezafibrate, and gemfibrozil on apoptosis, depolarization of mitochondrial membrane, and defective autophagy in BD lymphoblast cells. The viability of fibrate-treated BD lymphoblast cells increased to levels of normal lymphoblast cells. In addition, treatment with fibrates inhibited depolarization of mitochondrial membrane potential in BD lymphoblast cells. Defective autophagy in BD lymphoblast cells was normalized when treated with fibrates as indicated by increased acridine orange staining. The recovery of autophagy in BD lymphoblast cells is most likely attributed to the upregulation of autophagy proteins, lysosomal-associated membrane protein 1 (LAMP1), and LC3 I/II, after treatment with fibrates. This study therefore suggests that fibrates may have a therapeutic potential against BD.

Performance of different SNP panels for parentage testing in two East Asian cattle breeds

The International Society for Animal Genetics (ISAG) proposed a panel of single nucleotide polymorphisms (SNPs) for parentage testing in cattle (a core panel of 100 SNPs and an additional list of 100 SNPs). However, markers specific to East Asian taurine cattle breeds were not included, and no information is available as to whether the ISAG panel performs adequately for these breeds. We tested ISAGs core (100 SNP) and full (200 SNP) panels on two East Asian taurine breeds: the Korean Hanwoo and the Japanese Wagyu, the latter from the Australian herd. Even though the power of exclusion was high at 0.99 for both ISAG panels, the core panel performed poorly with 3.01% false-positive assignments in the Hanwoo population and 3.57% in the Wagyu. The full ISAG panel identified all sire-offspring relations correctly in both populations with 0.02% of relations wrongly excluded in the Hanwoo population. Based on these results, we created and tested two population-specific marker panels: one for the Wagyu population, which showed no false-positive assignments with either 100 or 200 SNPs, and a second panel for the Hanwoo, which still had some false-positive assignments with 100 SNPs but no false positives using 200 SNPs. In conclusion, for parentage assignment in East Asian cattle breeds, only the full ISAG panel is adequate for parentage testing. If fewer markers should be used, it is advisable to use population-specific markers rather than the ISAG panel.

Exploring the Genetic Signature of Body Size in Yucatan Miniature Pig

Since being domesticated about 10,000–12,000 years ago, domestic pigs (Sus scrofa domesticus) have been selected for traits of economic importance, in particular large body size. However, Yucatan miniature pigs have been selected for small body size to withstand high temperature environment and for laboratory use. This renders the Yucatan miniature pig a valuable model for understanding the evolution of body size. We investigate the genetic signature for selection of body size in the Yucatan miniature pig. Phylogenetic distance of Yucatan miniature pig was compared to other large swine breeds (Yorkshire, Landrace, Duroc and wild boar). By estimating the XP-EHH statistic using re-sequencing data derived from 70 pigs, we were able to unravel the signatures of selection of body size. We found that both selections at the level of organism, and at the cellular level have occurred. Selection at the higher levels include feed intake, regulation of body weight and increase in mass while selection at the molecular level includes cell cycle and cell proliferation. Positively selected genes probed by XP-EHH may provide insight into the docile character and innate immunity as well as body size of Yucatan miniature pig.

An approach to identify SNPs in the gene encoding acetyl-CoA acetyltransferase-2 (ACAT-2) and their proposed role in metabolic processes in pig

The novel liver protein acetyl-CoA acetyltransferase-2 (ACAT2) is involved in the beta-oxidation and lipid metabolism. Its comprehensive relative expression, in silico non-synonymous single nucleotide polymorphism (nsSNP) analysis, as well as its annotation in terms of metabolic process with another protein from the same family, namely, acetyl-CoA acyltransferase-2 (ACAA2) was performed in Sus scrofa. This investigation was conducted to understand the most important nsSNPs of ACAT2 in terms of their effects on metabolic activities and protein conformation. The two most deleterious mutations at residues 122 (I to V) and 281 (R to H) were found in ACAT2. Validation of expression of genes in the laboratory also supported the idea of differential expression of ACAT2 and ACAA2 conceived through the in silico analysis. Analysis of the relative expression of ACAT2 and ACAA2 in the liver tissue of Jeju native pig showed that the former expressed significantly higher (P<0.05). Overall, the computational prediction supported by wet laboratory analysis suggests that ACAT2 might contribute more to metabolic processes than ACAA2 in swine. Further associations of SNPs in ACAT2 with production traits might guide efforts to improve growth performance in Jeju native pigs.

Characterization of recombinant scFv antibody reactive with an apical antigen of Eimeria acervulina

The chicken monoclonal antibody (mAb), 6D-12-G10, reacts with an apical complex protein at the anterior tip of E. acervulina sporozoites that inhibits parasite invasion in vitro. Because this mAb was produced at low amount from the original hybridoma cells, an scFv antibody was constructed by amplification of the corresponding VH and VL genes and expressed in E. coli. The scFv antibody was produced at a minimum of 7 mg l−1 and exhibited virtually identical antigen reactivity as the original mAb.