Rats are an important laboratory animal for biomedical research. Though rats have some physiology and genetic similarities to human, several technical issues such as delicate in vitro culture system and low survival rate after pronuclear microinjection have hindered the development of transgenic rat generation. Accordingly, in this study, to produce transgenic rat, we established transposon-mediated insertional mutagenesis by cytoplasmic microinjection. The sleeping beauty transposon (SB) and SB-transposase recognize the precise genome integration into a TA nucleotide by ‘cut-and-paste’ mechanism. It mediates stable integration and reliable long-term expression. DNA, 0.4ng/ul SB vector (IR/DR-EF1a-eGFP-2A-IL2-pA-IR/DR) and mRNA, 5ng/ul SB-transposase were injected to 1-cell stage embryo and one transgenic rat was generated after full-term gestation. To confirm the genome insertion, GFP was detected by PCR. Further, this method was applied to generate transgenic rats producing Cas9 protein. DNA, 0.4ng/ul SB vector (IR/DR-CAG-Cas9-2A-eGFP-pA-IR/DR) and mRNA, 5ng/ul SB-transposase were injected to 1-cell stage embryo. Some of the in vitro cultured embryos showed GFP positive at blastocyst stage and Cas9 sequence was detected by PCR. One stillbirth pup was born to date and genome PCR on Cas9 was positive. In summary, the SB transposon system could be a highly effective method that contribute to the production of transgenic rats. If the protocols will be optimized, we successfully generated efficiently transgenic rats for human models by SB system. This work was supported by BK21 PLUS Program for Creative Veterinary Science, the National Research Foundation of Korea (2017R1A2B3004972) and the Technology Development Program (S2566872) by MSS.

Brucella abortus is a well-known intracellular zoonotic pathogen. Despite significant research to understand the pathogenesis, underlying mechanisms of the bacterial infection are not yet understood. Thus, prevention and control of the B. abortus infection is problematic in animal and human. Therefore, several methods involving random mutation have been used to identify the mechanisms and provide a solution for control and prevention of Brucella infection. B. abortus mutants were generated by random insertion of a transposon, Ez-Tn5TM pMODTM-3 <R6Kγori/MCS> into a chromosome. Characteristics of these mutants were investigated using biochemical testing, growth features, determination of biovar, antibiotic resistance and detection of virulence factors, T4SS, PGK, and CGS. In biochemical testing, B. abortus mutants were categorized according to 7 groups with different condition of ILATk, SUTC, and ELLM. Different growth features were also observed between wild type and mutants. In addition, both B. abortus wild type and mutants were determined as biovar type 1 by biovar test. Three virulence factors, T4SS, PGK, and CGS were detected by PCR. Therefore, B. abortus mutants were characterized by analysis of phenotyping and it might be useful for further studies of known pathogenesis of B. abortus infection and for identification of diagnostic markers of brucellosis.

The companion cells of the Arabidopsis thaliana egg and sperm, the central and vegetative cells, undergo active DNA demethylation prior to fertilization. However, its biological significance, extent of conservation, and targeting preferences are not yet clear. We recently showed that localized demethylation of interspersed, small transposable elements is a common feature of A. thaliana companion cells. The DEMETER DNA glycosylase encodes active DNA demethylase activity and is required for seed production. DME-mediated DNA demethylation in the central cell is required to establish imprinted gene expression in the endosperm, and is considered a master regulator for plant gene imprinting. However, the similarity among DME targets in the central and vegetative cells, despite their different functions and developmental fates, suggests that establishment of genomic imprinting may not be the basal function of DME. Lack of DEMETER in vegetative cells causes reduced methylation of transposons in sperm. Our observation suggests that the primary function of companion cell demethylation is to reinforce transposon silencing in plant gametes.

We have identified ATTIRTA1 transposon, a kind of mariner-type DNA transposon from Brassica rapa genome. A total of 811 inverted-terminal repeat, ITR consisting of the both terminal on ATTIRTA1 transposon were found from B. rapa v1.1 sequence. Among them 616 ITR were paired by two in each transposon, indicating three quarters of the transposon exists in original form. Around 10 percentage of the transposon, 82 ITR was located in gene, expecially only in intron. Using these ATTRRTA1, we developed a display system modified from AFLP technique and applied for this system to analyze genetic diversity of Korea Brassica rapa core collection. The collection includes 220 accessions representing the different morphotypes and geographical origin. The analysis of population structure revealed five subgroups and the clustering patterns matched well with their morphological traits. ATTIRTA1 transposon display seems useful marker system for studying genetic relationships. Presently we have profiled the components and contents of glucosinolate in the core collection to analyze genome wide association. This collection will be helpful to identify agriculturally desirable traits from other supspecies.

1. 자포니카 벼 114 계통에 대해 다양성과 근연관계를 확인하고자 MITE 중에서 mPing family를 이용하여 MITE-TD 기법으로 분석하여 품종간의 다양성 정도를 산출한 결과 마커들의 PIC 값이 0.293~0.499 범위로 나타났다. 2. 두 개의 mPing primer와 selective primer인 BfaI+G 와 BfaI+C의 조합을 이용하였을 때, 공시계통인 114개의 자포니카 벼 전체를 구분할 수 있었다. 3. NTSYS-pc를 이용한 근연관계 분석 결과, 유사계수의 범위는 0.802에서 부터 0.081까지였고, 자포니카 벼 114 품종은 크게 5 개의 그룹으로 분류되었다. 4. 8 개의 MITE-AFLP marker 연관분석을 밀양 23호/합천앵미 3호 조합 RIL을 이용하여 실시한 결과, 이들은 염색체 l번, 2번, 4번, 5번, 7번 그리고 9번에 각각 위치함을 확인하였다.

Pax6는 진화적으로 잘 보존된 homeobox유전자 그룹의 하나로 배 발생기 동안 시공간적으로 제한되어 발현된다. 이 실험은 말라리아 매개모기인 Anopheles stephemi에서의 Pax6 발현을 서로 다른 분자환경 조건에서 조사해 보기 위해 트랜스포존의 하나인 piggyBac과 Pax6에 결합하는 3xp3-EGFP를 사용한 생식세포 형질전환 방법을 사용하였다. 4개의 형질 전환 계열이 만들어졌고 형질전환율은 6.7%였으며, 도입 유전자는 여러 세

본 연구실에서는 이전의 실험에서 suppression subtractive hybridization(SSH)을 통하여 생쥐의 생후 1일자 난소와 5일자 난소에서 차이 나게 발현하는 유전자들의 목록을 얻었고 그 중에서 MT transposon-like element, clone MTi7(MTi7)이 성장하는 난포에서 더 높게 발현한다는 것을 알아냈다(Park et al., 2002). In situ hybridization과 RNA interference