Application of Recombinant Monoclonal Antibodies from Transgenic Chicken Bioreactors in Enzyme-Linked Immunosorbent Assay.

Transgenic chicken bioreactors can efficiently produce egg whites containing large quantities of recombinant proteins. We previously developed transgenic chickens that produce recombinant monoclonal antibodies (mAbs) against epidermal growth factor receptor 2 (HER2). However, the practical applications of mAbs derived from transgenic eggs have not yet been examined.

Production and characterization of eggs from hens with ovomucoid gene mutation.

Ovomucoid is a major egg white protein which is considered as the most dominant allergen in chicken eggs. Owing to the difficulty of separating ovomucoid from egg whites, researchers have adopted genetic deletion for development of hypoallergenic eggs. Previously, we used CRISPR/Cas9 to establish chickens with ovomucoid gene (OVM) mutations, but it remained unknown whether such hens could produce eggs at maturity.

Production of Recombinant Monoclonal Antibodies in the Egg White of Gene-Targeted Transgenic Chickens.

Increased commercial demand for monoclonal antibodies (mAbs) has resulted in the urgent need to establish efficient production systems. We previously developed a transgenic chicken bioreactor system that effectively produced human cytokines in egg whites using genome-edited transgenic chickens. Here, we describe the application of this system to mAb production.

Efficient production of human interferon beta in the white of eggs from ovalbumin gene-targeted hens.

Transgenic chickens could potentially serve as bioreactors for commercial production of recombinant proteins in egg white. Many transgenic chickens have been generated by randomly integrating viral vectors into their genomes, but transgene expression has proved insufficient and/or limited to the initial cohort. Herein, we demonstrate the feasibility of integrating human interferon beta (hIFN-β) into the chicken ovalbumin locus and producing hIFN-β in egg white.

Targeted mutagenesis in chicken using CRISPR/Cas9 system.

The CRISPR/Cas9 system is a simple and powerful tool for genome editing in various organisms including livestock animals. However, the system has not been applied to poultry because of the difficulty in accessing their zygotes. Here we report the implementation of CRISPR/Cas9-mediated gene targeting in chickens.

Chicken stem cell factor enhances primordial germ cell proliferation cooperatively with fibroblast growth factor 2.

An in vitro culture system of chicken primordial germ cells (PGCs) has been recently developed, but the growth factor involved in the proliferation of PGCs is largely unknown. In the present study, we investigated the growth effects of chicken stem cell factor (chSCF) on the in vitro proliferation of chicken PGCs. We established two feeder cell lines (buffalo rat liver cells; BRL cells) that stably express the putative secreted form of chSCF (chSCF1-BRL) and membrane bound form of chSCF (chSCF2-BRL).

Sall4-Gli3 system in early limb progenitors is essential for the development of limb skeletal elements.

Limb skeletal elements originate from the limb progenitor cells, which undergo expansion and patterning to develop each skeletal element. Posterior-distal skeletal elements, such as the ulna/fibula and posterior digits develop in a Sonic hedgehog (Shh)-dependent manner. However, it is poorly understood how anterior-proximal elements, such as the humerus/femur, the radius/tibia and the anterior digits, are developed.

Migration of cardiomyocytes is essential for heart regeneration in zebrafish.

Adult zebrafish possess a significant ability to regenerate injured heart tissue through proliferation of pre-existing cardiomyocytes, which contrasts with the inability of mammals to do so after the immediate postnatal period. Zebrafish therefore provide a model system in which to study how an injured heart can be repaired. However, it remains unknown what important processes cardiomyocytes are involved in other than partial de-differentiation and proliferation.

HMGB factors are required for posterior digit development through integrating signaling pathway activities.

The chromatin factors Hmgb1 and Hmgb2 have critical roles in cellular processes, including transcription and DNA modification. To identify the function of Hmgb genes in embryonic development, we generated double mutants of Hmgb1;Hmgb2 in mice. While double null embryos arrest at E9.

Cre-LoxP-regulated expression of monoclonal antibodies driven by an ovalbumin promoter in primary oviduct cells.

Background: A promoter capable of driving high-level transgene expression in oviduct cells is important for developing transgenic chickens capable of producing therapeutic proteins, including monoclonal antibodies (mAbs), in the whites of laid eggs. Ovalbumin promoters can be used as oviduct-specific regulatory sequences in transgenic chickens, but their promoter activities are not high, according to previous reports.

Results: In this study, while using a previously characterized ovalbumin promoter, we attempted to improve the expression level of mAbs using a Cre/loxP-mediated conditional excision system.

Improvement of transfection efficiency in cultured chicken primordial germ cells by percoll density gradient centrifugation.

Chicken primordial germ cells (PGCs) differentiate into germ cells in gonads. Because PGCs can be cloned and cultured maintaining germline competency, they are a good means of modifying the chicken genome, but the efficiency of plasmid transfection into PGCs is very low. In this study, I attempted to improve the efficiency of PGC transfection.

Ror-family receptor tyrosine kinases in noncanonical Wnt signaling: their implications in developmental morphogenesis and human diseases.

The Ror-family receptor tyrosine kinases (RTKs) play crucial roles in the development of various organs and tissues. In mammals, Ror2, a member of the Ror-family RTKs, has been shown to act as a receptor or coreceptor for Wnt5a to mediate noncanonical Wnt signaling. Ror2- and Wnt5a-deficient mice exhibit similar abnormalities during developmental morphogenesis, reflecting their defects in convergent extension movements and planar cell polarity, characteristic features mediated by noncanonical Wnt signaling.

HIV type 1 genetic diversity in Moyale, Mandera, and Turkana based on env-C2-V3 sequences.

The genetic diversity of HIV-1 subtypes circulating in three districts of northern Kenya, i.e., Turkana, Mandera, and Moyale, was studied.

The forkhead protein Foxj1 specifies node-like cilia in Xenopus and zebrafish embryos.

It has been proposed that ciliated cells that produce a leftward fluid flow mediate left-right patterning in many vertebrate embryos. The cilia on these cells combine features of primary sensory and motile cilia, but how this cilia subtype is specified is unknown. We address this issue by analyzing the Xenopus and zebrafish homologs of Foxj1, a forkhead transcription factor necessary for ciliogenesis in multiciliated cells of the mouse.

Recent diversity of human immunodeficiency virus type 1 in individuals who visited sexually transmitted infection-related clinics in Osaka, Japan.

By human immunodeficiency virus type 1 (HIV-1) antibody screening of people who visited sexually transmitted infection (STI)-related clinics (venereology, urology, and gynecology) and were considered to conduct high-risk sexual activities for HIV-1 infection in Osaka, Japan, during 1992 to 2004, a total of 54 HIV-1 infected individuals (51 Japanese males and 3 non-Japanese females) were identified. Based on the sequencing at env-C2V3 and pol regions, Japanese males were mostly of subtype B (50/51 cases), with the one remaining case being a recombinant circulating form, CRF01_AE, while 3/3 viruses in non-Japanese females were of CRF01_AE. Analysis of subtype B cases since 2001 showed that these viruses became wider in their genetic variation, including amino acid insertions and also deletions, than that of the cases before 2000.

miles-apart-Mediated regulation of cell-fibronectin interaction and myocardial migration in zebrafish.

The migration of myocardial precursor cells towards the embryonic midline underlies the formation of the heart tube and is a key process of heart organogenesis. The zebrafish mutation miles-apart (mil), which affects the gene encoding a sphingosine-1-phosphate receptor, is characterized by defective migration of myocardial precursor cells and results in the formation of two laterally positioned hearts, a condition known as cardia bifida. The mechanism that disrupts myocardial migration in mil mutants remains largely unclear.

Regulation of primary cilia formation and left-right patterning in zebrafish by a noncanonical Wnt signaling mediator, duboraya.

Primary cilia are microtubule-based organelles that project from the surface of nearly every animal cell. Although important functions of primary cilia in morphogenesis and tissue homeostasis have been identified, the mechanisms that control the formation of primary cilia are not understood. Here we characterize a zebrafish gene, termed duboraya (dub), that is essential for ciliogenesis.

Intrinsic kinase activity and SQ/TQ domain of Chk2 kinase as well as N-terminal domain of Wip1 phosphatase are required for regulation of Chk2 by Wip1.

The anti-oncogenic Chk2 kinase plays a crucial role in DNA damage-induced cell cycle checkpoint regulation. Recently, we have shown that Chk2 associates with the oncogenic Wip1 (PPM1D) phosphatase and that Wip1 acts as a negative regulator of Chk2 during DNA damage response by dephosphorylating phosphorylated Thr-68 in activated Chk2 (Fujimoto, H., Onishi, N.

Tuberculosis and oral Candida species surveillance in HIV infected individuals in Northern Kenya, and the implications on tuberculin skin test screening for DOPT-P.

Objective: To determine the pattern of opportunistic infections such as TB and Candida species in HIV infected patients in Northern Kenya.

Design: Cross-sectional study.

Setting: Five health facilities in Moyale (n=224), Mandera (n=121) and Turkana Kakuma; (n=83), Lopiding; (n=94) districts during different periods in 2003.

Regulation of the antioncogenic Chk2 kinase by the oncogenic Wip1 phosphatase.

The antioncogenic Chk2 kinase plays a crucial role in DNA damage-induced cell-cycle checkpoint regulation. Here we show that Chk2 associates with the oncogenic protein Wip1 (wild-type p53-inducible phosphatase 1) (PPM1D), a p53-inducible protein phosphatase. Phosphorylation of Chk2 at threonine68 (Thr68), a critical event for Chk2 activation, which is normally induced by DNA damage or overexpression of Chk2, is inhibited by expression of wild-type (WT), but not a phosphatase-deficient mutant (D314A) of Wip1 in cultured cells.

HIV type 1 subtypes in circulation in northern Kenya.

The genetic subtypes of HIV-1 circulating in northern Kenya have not been characterized. Here we report the partial sequencing and analysis of samples collected in the years 2003 and 2004 from 72 HIV-1-positive patients in northern Kenya, which borders Ethiopia, Somalia, and Sudan. From the analysis of partial env sequences, it was determined that 50% were subtype A, 39% subtype C, and 11% subtype D.

Modulation of GDF5/BRI-b signalling through interaction with the tyrosine kinase receptor Ror2.

The brachydactylies are a group of inherited disorders of the hands characterized by shortened digits. Mutations in the tyrosine kinase receptor Ror2 cause brachydactyly type B (BDB). Mutations in GDF5, a member of the BMP/TGF-beta ligand family, cause brachydactyly type C (BDC) whereas mutations in the receptor for GDF5, BRI-b, cause brachydactyly type A2 (BDA2).

The receptor tyrosine kinase Ror2 associates with and is activated by casein kinase Iepsilon.

Ror2, a member of the mammalian Ror family of receptor tyrosine kinases, plays important roles in developmental morphogenesis, although the mechanism underlying activation of Ror2 remains largely elusive. We show that when expressed in mammalian cells, Ror2 associates with casein kinase Iepsilon (CKIepsilon), a crucial regulator of Wnt signaling. This association occurs primarily via the cytoplasmic C-terminal proline-rich domain of Ror2.

Regulation of Chk2 gene expression in lymphoid malignancies: involvement of epigenetic mechanisms in Hodgkin's lymphoma cell lines.

The tumor suppressor Chk2 kinase plays crucial roles in regulating cell-cycle checkpoints and apoptosis following DNA damage. We investigated the expression levels of the genes encoding Chk2 and several cell-cycle regulators in nine cell lines from lymphoid malignancies, including three Hodgkin's lymphoma (HL) lines. We found that all HL cell lines exhibited a drastic reduction in Chk2 expression without any apparent mutation of the Chk2 gene.