피부를 대상으로 한 살균을 목적으로 하는 외용소독제 의 경우 식품 취급자에 오염된 미생물의 사멸 또는 제거 를 목적으로 활용될 수 있으며, 최근 개인위생에 대한 관 심 증가에 따라 제품 소비 증가와 제품 다양화가 두드러 지게 나타나고 있다. 살균 효능은 소독제의 핵심 품질 평 가 요소로서 수행 절차 및 조건에 따라 상이한 결과가 나 타날 수 있기 때문에 시험법의 효율성과 정확성을 높이기 위한 연구가 필요하다. 이에 본 총설논문에서는 주요 제 형별(겔형, 액제형, 와이프형) 시험법 개발 현황을 파악하 고 시험법별 특장점 분석 결과와 최근 관련 연구를 통하 여 제시된 시사점을 기반으로 향후 효능 평가 체계의 발 전 방향을 제시하고자 하였다. 인체 대상 시험법의 경우 시험 유형에 따라 소독제를 시험 대상 피부 표면에 처리 하는 조건이 다양화되어 있어 시험법 간 동등성에 대한 평가를 통해 소독제 제품의 성분이나 특성에 따라 최적의 시험 유형을 파악하고 그에 대응되는 적절한 평가 체계 및 관련 규제의 표준화의 필요성을 시사하였다. 특히 와 이프형 소독제의 경우 처리 방식이 미생물 제거 및 살균 에 직접적으로 영향을 미침에도 불구하고 피부에 노출하 는 손 대상 처리를 위한 사용 패턴의 표준화 사례가 부족 하였다. 한편 [전처리 - 소독제 노출 - 미생물 회수] 등 각 시험 절차별로 결과에 영향을 미치는 주요 결정 요인을 발굴하는 연구의 지속 수행을 통해 기존 시험법을 개선하고 신규 시험법을 개발하고자 하는 노력이 요구된다. 최 근 활발하게 개발되고 있는 ex vivo 시험법은 인체 시험 의 제한적인 연구 재현성과 같은 한계를 극복하면서도 인 간 피부 환경을 구현하기 위한 기술의 적용을 통해 연구 결과의 신뢰도를 확보할 수 있을 것으로 판단된다. 한편 손 피부를 대상으로 한 균총 연구 등 소독제 처리 전후 미생물의 특성과 분포 분석 관련 연구가 최근 다수 보고 되고 있어 이를 활용한 미생물 군집 단위의 소독제 효능 평가 시험법의 확립이 기대된다. 본 연구를 통해 제시된 소독제 효능 시험법의 현황 기반 발전 전략은 보다 효과 적인 개인위생 관리 확립을 통해 손을 통해 교차 오염되 는 미생물에 의한 감염성 질병 발생을 최소화하여 공중보 건 및 식품 안전성 향상에 기여할 수 있다.

The Transgenic livestock can be useful for the production of disease-resistant animals, pigs for xenotranplantation, animal bioreactor for therapeutic recombinant proteins and disease model animals. Previously, conventional methods without using artificial nuclease-dependent DNA cleavage system were used to produce such transgenic livestock, but their efficiency is known to be low. In the last decade, the development of artificial nucleases such as zinc-finger necleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas has led to more efficient production of knock-out and knock-in transgenic livestock. However, production of knock-in livestock is poor. In mouse, genetically modified mice are produced by co-injecting a pair of knock-in vector, which is a donor DNA, with a artificial nuclease in a pronuclear fertilized egg, but not in livestock. Gene targeting efficiency has been increased with the use of artificial nucleases, but the knock-in efficiency is still low in livestock. In many research now, somatic cell nuclear transfer (SCNT) methods used after selection of cell transfected with artificial nuclease for production of transgenic livestock. In particular, it is necessary to develop a system capable of producing transgenic livestock more efficiently by co-injection of artificial nuclease and knock-in vectors into fertilized eggs.

This study aimed to produce high-quality blastocysts and establish appropriate microinjection conditions for the introduction of target gene. First, we identified embryo development to the blastocyst stage after microinjection using the CRISPR/Cas9 system on the Cas9 protein or mRNA. As a result, we confirmed that blastocyst development in the Cas9 mRNA injected group significantly increased when compared to the Cas9 protein injected group (p<0.05). However, the blastocyst gene targeting rate increased in the Cas9 protein injected group when compared to the Cas9 mRNA injected group (p<0.05). Next, we treated the injection medium with 10 μg/ml of cytochalasin B (CB), and the microinjected embryos were cultured in CR1-aa medium supplemented with 0.1 μM of melatonin (Mela). Consequently, the blastocyst formation rate significantly increased in the CB treated group (p<0.05). After microinjecting embryos with the CB treated injection medium, we investigated blastocyst formation and quality via Mela treatment. Consequently, the Mela treated group demonstrated significantly increased blastocyst formation rates when compared to the non-treated group (p<0.05). Furthermore, immunofluorescence assay using RAD51 (DNA repair detection protein) and H2AX139ph (DNA damage detection protein) showed an increase in RAD51 positive cells in Mela treated embryos. Therefore, we verified the improvement in knock-in efficiency in microinjected bovine embryos using Cas9 protein. These results also demonstrated that the positive effect of the CB and Mela treatments improved the embryonic developmental competence and blastocyst qualities in genetically-edited bovine embryos.

The production of therapeutic protein from transgenic domestic animal is the major technology of biotechnology. Insulin-like growth factor-1 (IGF-1) is known to play an important role in the growth of the animal. The objective of this study is construction of knock-in vector that bovine IGF-1 gene is inserted into the exon 7 locus of β-casein gene and expressed using the gene regulatory DNA sequence of bovine β-casein gene. The knock-in vector consists of 5’ arm region (1.02 kb), bIGF-1 cDNA, CMV-EGFP, and 3’ arm region (1.81 kb). To express bIGF-1 gene as transgene, the F2A sequence was fused to the 5’ terminal of bIGF-1 gene and inserted into exon 7 of the β-casein gene. As a result, the knock-in vector is confirmed that the amino acids are synthesized without termination from the β-casein exon 7 region to the bIGF-1 gene by DNA sequence. These knock-in vectors may help to create transgenic dairy cattle expressing bovine bIGF-1 protein in the mammary gland via the expression system of the bovine β-casein gene.

The knock-in efficiency in the fibroblast is very important to produce transgenic domestic animal using nuclear transfer. In this research, we constructed three kinds of different knock-in vectors to study the efficiency of knock-in depending on structure of knock-in vector with different size of homologous arm on the β-casein gene locus in the somatic cells; DT-A_cEndo Knock-in vector, DT-A_tEndo Knock-in vector I, and DT-A_tEndo Knock-in vector II. The knock-in vector consists of 4.8 kb or 1.06 kb of 5’ arm region and 1.8 kb or 0.64 kb of 3’ arm region, and neomycin resistance gene(neor) as a positive selection marker gene. The cEndo Knock-in vector had 4.8 kb and 1.8 kb homologous arm. The tEndo Knock-in vector I had 1.06 kb and 0.64 kb homologous arm and tEndo Knock-in vector II had 1.06 kb and 1.8 kb homologous arm. To express endostatin gene as transgene, the F2A sequence was fused to the 5’ terminal of endostatin gene and inserted into exon 7 of the β-casein gene. The knock-in vector and TALEN were introduced into the bovine fibroblast by electroporation. The knock-in efficiencies of cEndo, tEndo I, and tEndo II vector were 4.6%, 2.2% and 4.8%, respectively. These results indicated that size of 3’ arm in the knock-in vector is important for TALEN-mediated homologous recombination in the fibroblast. In conclusion, our knock-in system may help to create transgenic dairy cattle expressing human endostatin protein via the endogenous expression system of the bovine β-casein gene in the mammary gland.