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. Here, we have reported on eggs laid by OVM-targeted hens. Except for watery egg whites, the eggs had no evident abnormalities. Real-time PCR revealed alternative splicing of OVM mRNA in hens, but their expression was limited. Immunoblotting detected neither mature ovomucoid nor ovomucoid-truncated splicing variants in egg whites. Sixteen chicks hatched from 28 fertilized eggs laid by OVM-targeted hens, and fourteen of the sixteen chicks demonstrated healthy growth. Taken together, our results demonstrated that OVM knockout could almost completely eliminate ovomucoid from eggs, without abolishing fertility. Thus, the eggs developed in this study have potential as a hypoallergenic food source for most patients with egg allergies.

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. The genes encoding the heavy and light chains of humanized anti-HER2 mAb, linked by a 2A peptide sequence, were integrated into the chicken ovalbumin gene locus using a CRISPR/Cas9 protocol. The knock-in hens produced a fully assembled humanized mAb in their eggs. The mAb expression level in the egg white was 1.4-1.9 mg/mL, as determined by ELISA. Furthermore, the antigen binding affinity of the anti-HER2 mAb obtained was estimated to be equal to that of the therapeutic anti-HER2 mAb (trastuzumab). In addition, antigen-specific binding by the egg white mAb was demonstrated by immunofluorescence against HER2-positive and -negative cells. These results indicate that the chicken bioreactor system can efficiently produce mAbs with antigen binding capacity and can serve as an alternative production system for commercial mAbs.

'Protected DNA Probes' capable of strong hybridization without removal of base protecting groups.

We propose a new strategy called the 'Protected DNA Probes (PDP) method' in which appropriately protected bases selectively bind to the complementary bases without the removal of their base protecting groups. Previously, we reported that 4-N-acetylcytosine oligonucleotides (ac(4)C) exhibited a higher hybridization affinity for ssDNA than the unmodified oligonucleotides. For the PDP strategy, we created a modified adenine base and synthesized an N-acylated deoxyadenosine mimic having 6-N-acetyl-8-aza-7-deazaadenine (ac(6)az(8)c(7)A). It was found that PDP containing ac(4)C and ac(6)az(8)c(7)A exhibited higher affinity for the complementary ssDNA than the corresponding unmodified DNA probes and showed similar base recognition ability. Moreover, it should be noted that this PDP strategy could guarantee highly efficient synthesis of DNA probes on controlled pore glass (CPG) with high purity and thereby could eliminate the time-consuming procedures for isolating DNA probes. This strategy could also avoid undesired base-mediated elimination of DNA probes from CPG under basic conditions such as concentrated ammonia solution prescribed for removal of base protecting groups in the previous standard approach. Here, several successful applications of this strategy to single nucleotide polymorphism detection are also described in detail using PDPs immobilized on glass plates and those prepared on CPG plates, suggesting its potential usefulness.

Prehistoric Sado Island populations of Sus scrofa distinguished from contemporary Japanese wild boar by ancient mitochondrial DNA.

Ancient mitochondrial DNA (mtDNA) mainly from Jomon Period Sus scrofa bone specimens (6,100-1,700 years old) was examined to clarify the genetic relationships between prehistoric and contemporary S. scrofa on Hokkaido, Honshu, Sado, and Izu islands of the Japanese Archipelago. Phylogenetic analysis of the mtDNA control region (574 bp) and analysis of pairwise nucleotide differences between prehistoric and contemporary S. scrofa sequences showed the following relationships between these groups: (1) a group genetically similar to contemporary Japanese wild boars was found mainly on Honshu Island, Hokkaido Island, and the Izu Islands, and (2) a monophyletic group distinct from contemporary Japanese wild boars was found on Sado Island. These results suggest that prehistoric people introduced S. scrofa from Honshu Island to Hokkaido Island and the Izu Islands. The estimated divergence times between the prehistoric Sado group and the other prehistoric S. scrofa is approximately congruent with the geological isolation of Sado Island from Honshu Island. Our results suggest that this extinct S. scrofa population was present on Sado Island as recently as around 2,000 years ago.

Phylogeography and population structure of the Japanese wild boar Sus scrofa leucomystax: mitochondrial DNA variation.

Phylogeographic characteristics and population structure of Japanese wild boar (Sus scrofa leucomystax) were investigated using mitochondrial DNA (mtDNA) sequence data. Sixteen Japanese wild boar haplotypes detected from partial sequences of the mtDNA control region (574-bp) from 180 Japanese wild boar specimens from 10 local populations on Honshu, Shikoku, and Kyushu islands and 41 haplotypes from other S. scrofa were analyzed using the neighbor-joining method. The Japanese wild boars were more closely related to Northeast Asian wild boars from Mongolia than to the other Asian continental S. scrofa. The Japanese and Northeast Asian wild boars were not significantly distinguished by corrected average pairwise difference analysis. The ancestors of Japanese wild boars are suggested to have been part of the continental S. scrofa population that spread from Southeast to Northeast Asia during the Middle to Late Pleistocene. The Japanese wild boar mtDNA haplotype cladogram shows 95% parsimoniously plausible branch connections supporting three sympatric clades. Nested clade analysis indicates that these three clades are the result of distinct historical events or gene flow. The present population of Japanese wild boars may have been formed by a few independent migrations of distinct clades from the continent with subsequent mixing on the Japanese Islands.

Variation in mitochondrial DNA of Vietnamese pigs: relationships with Asian domestic pigs and Ryukyu wild boars.

Mitochondrial DNA (mtDNA) sequences (574 bp) of 30 Vietnamese pigs (large and small) were examined and compared with those of 61 haplotypes from wild boars and domestic pigs from various locations in Asia. The large Vietnamese pigs had genetic links to Ryukyu wild boars in southern Japan. The small Vietnamese pigs were closely related to other East Asian domestic pigs. These results indicate that Vietnamese pigs are genetically diverse and may be descendents of wild and domestic pigs from other regions of Asia.

Prehistoric introduction of domestic pigs onto the Okinawa Islands: ancient mitochondrial DNA evidence.

Ancient DNAs of Sus scrofa specimens excavated from archaeological sites on the Okinawa islands were examined to clarify the genetic relationships among prehistoric Sus scrofa, modern wild boars and domestic pigs inhabiting the Ryukyu archipelago, the Japanese islands, and the Asian continent. We extracted remain DNA from 161 bone specimens excavated from 12 archaeological sites on the Okinawa islands and successfully amplified mitochondrial DNA control region fragments from 33 of 161 specimens. Pairwise difference between prehistoric and modern S. scrofa nucleotide sequences showed that haplotypes of the East Asian domestic pig lineage were found from archaeological specimens together with Ryukyu wild boars native to the Ryukyu archipelago. Phylogenetic analysis of 14 ancient sequences (11 haplotypes; 574 bp) indicated that S. scrofa specimens from two Yayoi-Heian sites (Kitahara and Ara shellmiddens) and two Recent Times sites (Wakuta Kiln and Kiyuna sites) are grouped with modern East Asian domestic pigs. Sus scrofa specimens from Shimizu shellmidden (Yayoi-Heian Period) were very closely related to modern Sus scrofa riukiuanus but had a unique nucleotide insertion, indicating that the population is genetically distinct from the lineage of modern Ryukyu wild boars. This genetic evidence suggests that domestic pigs from the Asian continent were introduced to the Okinawa islands in the early Yayoi-Heian period (1700-2000 BP), or earlier.