The honey bee, Apis mellifera, has a defense system, including detoxification, antioxidation, and immunity pathways, against external stimulation such as chemicals, stress, and pathogens. However, pesticides, particularly neonicotinoids and butenolids, have been recently reported to alter physiological changes in honey bee. In this study, we investigated the expression levels of eight genes categorized into detoxification (CYPQ3), antioxidation (CAT and SOD2), and immune system (Abaecin, Apidaecin, Defensin1, Defensin2, and Hymenoptaecin), in five tissues (Head, Thorax, Gut, Fat body, and Carcass) of honey bee treated with three pesticides (Acetamiprid, Imidacloprid, and Flupyradifurone) using quantitative real-time PCR. Gene expression patterns was varied depending on the type of pesticides and tissues. However, among eight genes, the expression levels of CYPQ3 was notably induced, but those of AMPs were generally reduced by all pesticides tested in this study in five tissues. These suggest that CYPQ3-mediated detoxification pathway is induced, but AMP-mediated immune system might be disrupted when honey bee is exposed to neonicotinoids and butenolid.

The specific genetic modification in porcine somatic cells by gene targeting has been very difficult because of low efficiency of homologous recombination. To improve gene targeting, we designed three kinds of knock-out vectors with α1,3-galactosyltransferase gene (α1,3-GT gene), DT-A/pGT5’/neo/pGT3’, DT-A/NLS/pGT5’/neo/pGT3’ and pGT5’/neo/ pGT3’/NLS. The knock-out vectors consisted of a 4.8-kb fragment as the 5’ recombination arm (pGT5’) and a 1.9-kb fragment as the 3’ recombination arm (pGT3’). We used the neomycin resistance gene (neo) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. These vectors have a neo gene insertion in exon 9 for inactivation of α1,3-GT locus. DT-A/pGT5’/neo/pGT3’ vector contain only positive-nega-tive selection marker with conventional targeting vector. DT-A/NLS/pGT5’/neo/pGT3’ vector contain positive-negative selection marker and NLS sequences in upstream of 5’ recombination arm which enhances nuclear transport of foreign DNA into bovine somatic cells. pGT5’/neo/pGT3’/NLS vector contain only positive selection marker and NLS sequence in downstream of 3’ recombination arm, not contain negative selectable marker. For transfection, linearzed vectors were introduced into porcine ear fibroblasts by electroporation. After 48 hours, the transfected cells were selected with 300 μg/ml G418 during 12 day. The G418-resistant colonies were picked, of which 5 colonies were positive for α1,3-GT gene disruption in 3´ PCR and southern blot screening. Three knock-out somatic cells were obtained from DT-A/NLS/ pGT5’/neo/pGT3’ knock-out vector. Thus, these data indicate that gene targeting vector using nuclear localization signal and negative selection marker improve targeting efficiency in porcine somatic cells.

Gene targeting is a genetic technique that utilizes homologous recombination between an engineered exogenous DNA fragments with the endogenous genome of an organism. In domestic animal, gene targeting has provided an important tool for producing Knock-out pig for GGTA1 gene to use xenotransplantation. The frequency of homologous recombination is a critical parameter for the success of gene targeting. The efficiency of homologous recombination in somatic cells is lower than that in mouse ES cells. So the application of gene targeting in somatic cells has been limited by its low efficiency. Recently, knock-out rat and mouse was generated by introducing nonhomologous end joining (NHE)-mediated deletion or insertion at the target site using zinc-finger nucleases (ZFN). Therefore, the development of effective knock-out and knock-in techniques in domestic animal is very important in biomedical research. In this present study, we investigated whether homologous recombination events occurs at cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene locus using ZFN in porcine primary fibroblast. CMAH-targeted ZFN DNA and mRNA were purchased from SIGMA-Aldrich. CMAH neo targeting vector consists of the neomycin resistance gene as a positive selectable marker gene, 789 bp 5’ arm and 763 bp 3’ arm from Exon 8 of CMAH gene. For transfection, the targeting vector and ZFN DNA or mRNA were introduced into ear fibroblasts cells of Chicago miniature pig by electroporation. After selection of G-418, PCR analysis was performed using 213 colonies transfected with ZFN DNA or mRNA. As a result, 39 positive colonies were identified in colonies transfected with ZFN DNA or mRNA. To our knowledge, this study provides the first evidence that the efficiency of gene targeting using ZFN was higher than that of conventional gene targeting in the porcine fibroblast. These cell lines may be used in the production of CMAH knock-out for xenotransplantation.

Dentin, a major component of teeth, is formed by odontoblasts which produce the dentin matrix beneath the dental epithelium and induce the mineralization of dentin. To date, the biochemical properties of dentin matrix proteins have been well characterized, but upstream regulators of these proteins are not yet well known. Recently in this regard, several transcription factors have been identified as potential regulators of matrix proteins. Most transcription factors are generally involved in diverse biological processes and it is essential to identify those that are odontoblast-specific transactivators to further understand the process of dentin formation. We thus analyzed the expression pattern of dentin matrix proteins and the activities of established transactivators containing a Cre-locus. Expression analyses using in situ hybridization showed that dentin matrix proteins are sequentially expressed in differentiating odontoblasts, including type-I collagen, Dmp-1 and Dspp. The activities of the transactivators were evaluated using β-galactosidase following the generation of double transgenic mice with each transactivator and the ROSA26R reporter line. The β-galactosidase activity of each transactivator paralled the expression of the matrix proteins. These results thus showed that these transactivators could be utilized for odontoblastspecific conditional gene targeting. In addition, time- and tissue-specific conditional gene targeting might also be achieved using a combination of these transactivators. Odontoblast-specific conditional gene targeting with these transactivators will likely also provide new insights into the molecular mechanisms underlying dentin formation.

An increasing preference for good eating quality of rice among consumers has become one of the important considerations in rice breeding. Amylose content is a leading factor affecting eating quality of rice. Amylose composition is determined by the relative activity of soluble starch synthase (SSS) and granule-bound starch synthase (GBSS). This study focused on modifying the expression of SSSI gene which is responsible for amylopectin and amylose synthesis in rice by using RNA interference (RNAi) technology. The transgenic rice plants showed various amylose content in rice grains. Favorable rice lines were selected according to genomic PCR, transgene expression and amylose contents analysis. A semi-quantitative RT-PCR was carried out to determine the expression level of SSSI gene after flowering of transgenic rice and wild type. Down-regulation of SSSI gene in transgenic plants was evident in the decreasing expression in rice grains. Accordingly, scanning electron microscopy (SEM) analysis revealed uniform size with smooth curves starch granules in down-regulation rice lines, in contrast with the non-uniform granules in wild type. Results indicated that RNAi-SSSI transgenic lines produced low amylose contents that fell between glutinous and non-glutinous rice. This study showed that down-regulation of endogenous SSSI may improve the eating quality in rice.

Rice is one of the most important major food crops which provide the major food for more than half of global population. To improve the grain quality as well as grain yield has been the essential breeding goal in rice. The composition of amylopectin is the determinant of rice eating quality under certain threshold of protein content and the ratio of amylose and amylopectin. In this study, RBE 1 driven by CaMV-35S promoter was constructed and transformed using Agrobacterium tumefaciens. We selected single copy with low amylose content among transgenic lines. The mRNA expression was investigated using RT-PCR, and enzyme activity was determined using activity staining method in mid-milky stage endosperm. Also, the overexpression vectors for RBE 1 and SSS 1 driven by seed specific globulin promoter were constructed, respectively. Moreover, the RNA interference vectors for soluble starch synthase 1 and granule bound starch synthase 1 derived by CaMV35S promoter were constructed, respectively and transformed using Agrobacterium tumefaciens. The transgene has been confirmed by amplification of HPT and target gene. The transgenic plants obtained will be used to investigate the gene function of related starch pathway in plant cells using Gopumbyeo as a wild type rice, based on the gain-of-function and the loss-of-function. The development of designed site-specific endonucleases boosted the establishment of gene targeting (GT) techniques in a row of different species. However, the methods described in plants require a highly efficient transformation and regeneration procedure and, therefore, can be applied to very few species. Here, we describe a highly efficient GT system that is suitable for all transformable plants regardless of transformation efficiency. Efficient in planta GT was achieved in rice by expression of a site specific endonuclease (SSS1::ZFN) that not only cuts within the target but also the chromosomal transgenic donor, leading to an excised targeting vector.

배아줄기세포를 이용한 형질전환동물의 제조는 유전자의 기능 연구에 필수적이다. 특히 유전자 파괴 생쥐는 유전자의 기능 연구뿐만 아니라 사람 질병 연구에 중요한 모델이 되어 왔다. 유전자 적중법(gene targeting)과 유전자 함정법(gene trapping)은 ES 세포에서 녹아웃(knockout) 생쥐를 제조하는 대표적인 방법이다. 20여 년 전 유전자 적중법과 함정법이 최초로 개발된 이후에 이 기술은 많은 변화를 거쳤다. 특히 상동재조합에 기초한 전통적 유전자 적중법은 대량 제조기반의 조건부 유전자 적중법의 개발로 이어졌고, 유전자 적중법 및 유전자 함정법의 장점 요소의 조합은 유전자를 파괴하는 범위를 넓혔고, 유전자 적중을 더욱 효율적으로 만들었다. 이런 기술은 특정 유전자를 표적으로 하는 다양한 종류의 돌연변이 형질전환동물을 제조할 수 있게 하여 포스트게놈 시대에 요구되는 전체 유전체의 기능 연구를 더욱 효과적으로 진행시켜 줄 것이다.