Kazuyoshi Kaminaka

Production of Transgenic Chimera Rabbit Fetuses Using Somatic Cell Nuclear Transfer

We produced aggregate chimeric embryos between blastomeres from the somatic cell nuclear transfer (SCNT) embryos and blastomeres from normal embryos. The SCNT embryos were produced by fusing enucleated oocytes with GFP gene introduced fibroblast cells, which were derived from a day 16 fetus. GFP gene-introduced fibroblast cells were cultured and passaged four to 12 times over a period of 45-79 days before SCNT. After transferring them into pseudopregnant recipient rabbits, the 15-day postcoitus fetuses were collected. We examined the existence of the cells derived from SCNT embryos in the fetus stage of pregnancy to detect the GFP gene. Fetuses that were not collected continued to develop into newborn rabbits. Two hundred and thirty-six chimeric embryos were produced using 39 SCNT morula stage embryos, and these embryos were transferred to 11 recipient rabbits. As a result, 27 normally developed and 16 degenerated concepti were obtained. The GFP gene-positive signals were detected in one of the fetuses, two of the placentae, and two of the degenerated concepti. In this study, we found that the rabbit SCNT embryos have the ability to develop and differentiate in vivo. We also demonstrated a novel method of producing a transgenic rabbit using SCNT.

Amyloid β peptides with an additional cysteine residue can enhance immunogenicity and reduce the amyloid β burden in an Alzheimer’s disease mouse model

For the development of a safe vaccine for Alzheimers disease (AD), we studied the immunogenicity of amyloid beta (Abeta) peptides without adjuvant. Addition of a cysteine residue (Cys) to Abeta peptides enhanced immunogenicity in mice compared to those without Cys. Vaccination with the Abeta-Cys peptides reduced Abeta deposits in AD model mice. From these results, the Abeta-Cys peptides, administered without adjuvant, are considered candidates for vaccine therapy for AD.

Self-priming of hepatitis C virus RNA

Abstract To identify hepatitis C virus (HCV) infection, HCV genomic ribonucleic acid (RNA) can be detected using the reverse transcription-nested polymerase chain reaction (RT-nested PCR). HCV replication involves the production of a complementary, genomic-length, negative RNA strand via semiconservative RNA synthesis, utilizing negative strand specific reverse transcription and subsequent DNA synthesis. It is important to exclude the presence of self-priming in negative strand specific RT-nested PCR assay. In these experiments, HCV genomic RNA was subjected to reverse transcription without addition of a primer, and the resultant complementary deoxyribonucleic acid (cDNA) was produced. Digestion of template with RNase suggests that the template RNA is reverse transcribed with an RNA primer, not a DNA primer. Therefore, caution must be employed in interpreting studies of HCV replication using negative strand specific reverse transcription and PCR.

Influenza Virus M2e with Additional Cysteine Residues Shows Enhanced Immunogenicity and Protection Against Lethal Virus Challenge

The amino acid sequence of the extracellular domain of matrix protein 2 (M2e) is conserved among all subtypes of influenza A viruses. Therefore, the M2e peptide can be considered as a target antigen for the development of a universal influenza vaccine. We evaluated the effects of adding cysteine residues to a peptide of amino acids 2-24 of M2e. Mice immunized with some of these peptides containing one, two, three, four, or five extra cysteines displayed enhanced antibody titers to M2e. In addition, immunization with a peptide containing three extra cysteines, along with an aluminum adjuvant, protected mice more effectively against a lethal influenza virus challenge than the original M2e peptide. These results indicated that an M2e peptide containing additional cysteine residues could be a universal influenza vaccine candidate even without the addition of strong adjuvants.

Detection of antibody against nonstructural (NS) 5 region of hepatitis C virus polyprotein

Abstract A cDNA clone, C8-2, derived from nonstructural (NS) protein 5 region of hepatitis C virus (HCV) genome hasbeen isolated and was expressed in Escherichia coli as a fusion protein with β -galactosidase ( β -Gal). The recombinant protein, β -Gal/C8-2, was partially purified and used for detection of anti-HCV antibody by enzyme-linked immunosorbent assay (ELISA). Anti-C8-2 antibody was detected in 47% of patients with post-transfusion non-A, non-B (NANB) acute hepatitis and in 55% of patients with NANB chronic liver diseases. Of 1491 normal blood donors, ten (0.7%) were positive for anti-C8-2 antibody, and three of these ten samples were negative for anti-C100-3 antibody. HCV RNA was detected in one of these three samples. These results suggest that the recombinant protein containing C8-2 peptide may be useful as another nonstructural protein epitope for diagnosis of hepatitis C and screening of blood donors.