Preparation of knockout Mouse of a transregulatory fector, OCZF which is specifically expressed inosteoclasts

为了研究破骨细胞特异表达的转录调控因子OCZF（Osteoclast zinc finger protein）在破骨细胞中的作用，我们从小鼠BAG克隆ES细胞基因组文库中克隆了OCZF的小鼠同源物LRF（Leukocyterelated factor）基因组克隆，并制备了含有新霉素抗性基因的靶向载体。通过电穿孔将靶向载体导入ES细胞，并加入G418。经过9天的筛选，筛选出500个抗性ES克隆。对ES抗性细胞进行PCR和Southern分析，获得6个LRF（-/+）ES细胞克隆。我们进一步提高G418的浓度，获得LRF（-/-）ES细胞，分析LRF基因缺失情况下破骨细胞分化的可能性。我们决定与美国和日本的研究小组合作，采用LRF基因敲除小鼠，并在体外进行了OCZF/LRF基因的siRNA敲除。我们分析了RNA和DNA容易导入小鼠巨噬细胞系RAW 264的条件，发现使用Nucleofector，RAW 264细胞被RNA导入，高达80%的细胞群体。制备了几种小RNA干扰物，以抑制RAW 264细胞中LRF基因的表达。将RNAi导入经RANKL刺激的RAW 264细胞中显著抑制RAW 264细胞的破骨细胞分化。

To investigate the role of transcriptional regulator, OCZF(Osteoclast zinc finger protein) specifically expressed in osteoclasts, we isolated a genome clone of mouse homologue of OCZF, LRF(Leukocyte-related factor) from mouse BAG clone ES cell genome library and prepared targeted vector which had neomycin resistant gene. ES cells were introduced by targeted vector by electroporation and G418 was added. After selection for 9 days, 500 resistant ES clones were picked up. ES resistant cells were analyzed by PCR and southern analysis and 6 LRF (-/+) ES cell clones were obtained. We then increased the concentration of G418 and obtained LRF(-/-) ES cells to analyze the possibility of osteoclast differentiation in the absence of LRF gene.During the course of the experiments, we are informed that LRF knockout mouse are already prepared by American group and mouse is embryonic lethal. We then determined to collaborate with American group and Japanese group to use LRF knockout mouse.We also performed to knockout OCZF/LRF gene by using siRNA in vitro. We analyzed the condition which is easily introduced RNA and DNA into mouse macrophage cell line, RAW264 and found that RAW 264 cells were introduced by RNA up to 80 % of the cell population by using Nucleofector. Several small RNAis were prepared to repress the expression LRF gene in RAW 264 cells. Introduction of RNAi into RAW 264 cells stimulated with RANKL markedly inhibited osteoclast differentiation of RAW 264 cells.