Toru Sawasaki

Co-injection of restriction enzyme with foreign DNA into the pronucleus for elevating production efficiencies of transgenic animals

The microinjection method for production of transgenic farm animals requires specialized techniques and results in intolerably low production efficiencies. We investigated whether or not co-injection of foreign DNA constructs with restriction endonuclease into the pronucleus of mouse zygotes would improve the integration frequencies of foreign DNA into the host genome. Two kinds of DNA constructs that have no EcoRI site in their sequences were used for co-microinjection. With reference to the results of experiments in which EcoRI alone was injected at various amounts varying from 10(-9) to 10(-5) U/nucleus, the amount of 5x10(-8) U/nucleus that showed survival rate of 60.6% was used for the co-injection with DNA. Successful transgenesis of co-injected embryos was identified by DpnI-Bal31 digestion method for single embryos and by PCR method for pups born, respectively. The overall efficiency for the integration of foreign DNA in single embryos and live-born pups obtained by the co-injection procedures were 17.9% compared with 9.1% obtained by the injection of DNA alone. The results suggest that co-injection of foreign genes with restriction enzyme may elevate the integration rate of foreign genes into host genomes.

Equine inhibin/activin βA-subunit mRNA is expressed in the endometrial gland, but not in the trophoblast, during pregnancy

The expression of both inhibin α‐ and inhibin/activin βA‐subunit mRNA was examined in equine uteroplacental tissues collected during pregnancy (days 90 to 300). Northern blot analysis revealed that 5 transcripts (7.0, 4.1, 3.4, 2.6, 1.5 kb) of βA‐subunit were present, and the most abundantly expressed transcript was the 1.5 kb one. Relatively high levels of the 1.5 kb transcript were seen in the second trimester of pregnancy compared to what was found in the third trimester. To identify the tissue localization of βA‐subunit mRNA, in situ hybridization was performed, and the positive signal was observed exclusively in the endometrial glands, but not in the fetal placental tissue (trophoblast) at days 150, 210, and 300 of pregnancy. On the other hand, inhibin α‐subunit transcript could not be detected at any stage of pregnancy examined either by Northern blot analysis or in situ hybridization. Although the factor(s) regulating the gene expression of βA‐subunit in this equine tissue is currently unknown, these results suggest that activin, but not inhibin, is predominantly produced in the endometrial glands of the pregnant mare, and thus produced activin may play a paracrine or endocrine role during pregnancy in this species. Mol. Reprod. Dev. 47:363–369, 1997.

Correlation between spontaneous feeding behavior and neuropeptide Y profile in the third ventricular cerebrospinal fluid of goats.

Feeding behavior is regulated by neural signals in the hypothalamus, but secretory activities of these signals in vivo and their relationship with spontaneous feeding remain to be solved. In the present study, we investigated the correlation between neuropeptide Y (NPY) and somatostatin (SRIF) profiles in cerebrospinal fluid (CSF) and spontaneous feeding behavior in goats. CSF samples were collected every 15 min for 8 h from the third ventricle and feeding behavior was observed throughout the experimental period. The spontaneous feeding behavior, the mean duration of which was 58 min, occurred with an interval of 146 min. NPY in the CSF fluctuated in an episodic fashion with a 145 min interval. Each NPY episode was followed by spontaneous feeding with a time lag of 24 min. SRIF levels in CSF changed more frequently in a pulsatile manner and were related to neither NPY profiles nor feeding behavior. These results suggest that NPY, but not SRIF, is a physiological signal to drive feeding in goats.

Molecular cloning of cDNA encoding the c-kit receptor of Shiba goats and a novel alanine insertion specific to goats and sheep in the kinase insert region

The complete open reading frame (ORF) of the c-kit cDNA was cloned from a cerebellar cDNA library of the Shiba goat (Capra hircus var Shiba) with the dominant black-eyed white phenotype. The analysis of the deduced amino acid sequence revealed the presence of a single amino acid insertion (alanine) in the kinase insert (KI) region. While the newly found alanine insertion is not correlated with the coat color phenotype of goats, it appears to be characteristic of the c-kit genes in goats and sheep. Although the biological significance of the insert remains to be investigated, its phylogenetically limited distribution will provide us with a useful and interesting tool to analyze the problems of evolution of sheep and goats in bovidae.