follistatin-related protein : analysis of arthritis induced in its transgenic mice

为了获得在关节软骨中特异表达卵泡抑素相关蛋白（FRP）的转基因小鼠，我们构建了Col2a1-mFRP和Col2a1-FLAG-mFRP转基因小鼠。在这两种转基因中，小鼠FRP (mFRP) cDNA都位于II型胶原启动子的下游。在Col2a1-FLAG-mFRP中，对mFRP cDNA的5'核苷酸序列进行修饰，生成在N端带有FLAG表位标签的蛋白。为了确保转基因能够在II型胶原启动子的调控下转录，我们将Col2a1-FLAG-mFRP转移到发育阶段的小鼠畸胎瘤细胞系ATDC5至软骨细胞，并估计转录的mRNA。RT-PCR检测到FLAG-mFRP mRNA的特异引物。在本转基因小鼠实验中，为了便于FLAG表位标签检测基因产物，我们采用了这两种转基因的Col2a1-FLAG-mFRP。将Col2a1-FLAG-mFRP注射到DBA/1 （Crj）小鼠的胚胎中，将形态完整的胚胎移植到ICR小鼠的输卵管中。在注入转基因的344个胚胎中，有310个胚胎移植到11只老鼠身上，两只老鼠怀孕并生了4只后代。使用flag - mfrp特异性引物对基因组DNA进行RT-PCR分析，仅在一只F0小鼠中检测到转基因。该转基因阳性F0小鼠的表型与野生型小鼠几乎相同。我们现在通过将这种F0小鼠与野生DBA/1 （Crj）小鼠交配来生产F1小鼠，并对出生的53只F1小鼠进行转基因分析。作为对照研究，我们用胶原蛋白单克隆抗体（Arthritogenic mAb Cocktail， IBL）对野生型小鼠进行了关节炎诱导实验，并成功地使所有被试小鼠产生关节炎。今后，我们将继续对胚胎进行转基因注射，以便在本研究中获得更多的转基因小鼠候选品系。这个项目正在进行中。

To produce the transgenic mice, which can overexpress follistatin-related protein (FRP) specifically in the articular cartilage, the transgenes named Col2a1-mFRP and Col2a1-FLAG-mFRP were constructed. In both transgenes, mouse FRP (mFRP) cDNA was arranged downstream to the promoter of type II collagen. In Col2a1-FLAG-mFRP, the 5' nucleotide sequence of mFRP cDNA was modified to generate the protein bearing a FLAG epitope tag in the N terminus. To make sure that the transgenes could be transcribed under the regulation of type II collagen promoters, Col2a1-FLAG-mFRP was transferred to a mouse teratoma cell line ATDC5 in the developing stage to chondrocytes, and the transcribed mRNA was estimated. FLAG-mFRP mRNA was certainly detected by RT-PCR with its gene-specific primers. In this experiment producing transgenic mice, Col2a1-FLAG-mFRP of the two transgenes was adopted for the easy detection of the gene products by FLAG epitope tags. Col2a1-FLAG-mFRP was injected into embryos of DBA/1 (Crj) mice, and morphologically intact embryos were transferred to the uterine tubes of ICR mice. 310 embryos out of 344 transgene-injected ones were transplanted to eleven mice, and two mice became pregnant and bore four F0 offspring. RT-PCR analysis of genomic DNA with FLAG-mFRP-specific primers detected the transgenes in only one F0 mouse. This transgene-positive F0 mouse had almost the same phenotype as the wild type mouse. We are now producing F1 mice by mating this F0 mouse with wild DBA/1 (Crj) mice, and analyzing the transgenes in the born 53 F1 mice. As a control study, we performed arthritis-induction experiments in wild type mice with monoclonal antibodies to collagen (Arthritogenic mAb Cocktail, IBL), and succeeded in causing arthritis in all the tested mice. From now on, we will continue the transgene injection to the embryos in order to get more candidate strains of transgenic mice in this study. This project is now under way.