修饰mRNA法建立大黄鱼诱导多能干细胞

Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cell artificially derived from a non-pluripotent cell - typically an adult somatic cell - by inducing a "forced" expression of specific genes. iPSCs was originally achieved by Yamanaka and colleagues by enforced expression of four transcription factors, KLF4,c-MYC, OCT4, and SOX2 (KMOS), by using retroviral vectors. Up to date, iPSCs have made some achievements in mammals, but application of iPSCs is limited by the low efficiency of iPSC derivation and the fact of higher tumorigenicity that most protocols modify the genome to effect cellular reprogramming. Currently, a simple, nonintegrating approach for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses has been established. The large yellow croaker (Larimichthys crocea) is one of the most economically important marine fish species in China. With the rapid development of the aquaculture industry, however, lower growth rates, earlier sexuality maturity and weaker disease resistance became prevalent in cultured stocks of large yellow croaker. In the project, we will clone full length cDNAs of four transcription factors, KLF4,c-MYC, OCT4, and SOX2 (KMOS) from large yellow croaker based on EST database from our group and their conserved sequences from other animals. The temporal and spatial expression of KMOS in various tissues and different embryonic development stages will be studied by using Real-time PCR and in situ hybridization. Based on the above results, RNA transfected kidney cell line of large yellow croaker will be set up with RNAiMAX or TransITmRNA cationic lipid delivery vehicles by using a combination of RNA modifications and a soluble interferon inhibitor. iPSCs of large yellow croaker will be established by using a popular reprogramming regimen. A number of molecular and functional assays will be performed to further assess whether the RiPSCs had been reprogrammed to pluripotency. Results of this project will represent a safe, efficient strategy for somatic cell reprogramming and directing cell fate in fish. It will have broad applicability for basic research, disease model, drug screening model and regenerative medicine in fish.

iPS细胞技术在哺乳动物的研究中已取得一定成果，但基于DNA的诱导方法存在着效率低、致瘤性高等缺点。本项目将以我国重要的海水养殖鱼类-大黄鱼为实验材料，根据本课题组大黄鱼EST数据库所获得的Oct4、Sox2、Klf4和c-Myc 4个转录因子的基因片段并结合其他动物4个基因的保守序列设计引物，通过RT-PCR、RACE等分子生物学方法获得大黄鱼的Oct4、Sox2、Klf4和c-Myc基因的全长cDNA，结合QRT-PCR和原位杂交分析这些基因在各组织和胚胎发育过程中的时空表达特点，然后采用基于RNA的iPS细胞技术,通过导入修饰过的4个基因的mRNA到大黄鱼体细胞中，将其诱导为iPS细胞并进行鉴定和培养，进而建立一种安全、高效、快速的大黄鱼多能干细胞诱导方法，为研究细胞重编程的机理、建立个体特异的多能干细胞系奠定基础，为从根本上解决大黄鱼养殖过程中出现的各种问题提供新思路。

本研究对大黄鱼多能性基因的克隆与表达、mRNA修饰及体外转录、组织原代细胞的培养、基因转染原代体细胞及iPS细胞鉴别等进行了一系列研究，有望通过iPSCs技术，建立个体特异的多能干细胞系，为大黄鱼生长发育、种质资源保护、疾病防治及药物筛选研究等提供宝贵的实验材料，是一条新的从根本上解决大黄鱼各种养殖难题的有效策略模式。主要研究成果如下：. 1. 多能性基因的克隆与表达分析. 采用SMART-RACE技术克隆获得了大黄鱼Oct4、Sox2、Klf4和cMyc 4个多能性基因cDNA全长并进行了生物信息学分析； 采用实时荧光定量PCR（qRT-PCR）技术和原位杂交技术研究了4个多能性基因在组织和胚胎中的时空表达模式并进行了比较，还对其功能进行了初步分析。. 2. 修饰mRNA介导的iPS细胞诱导体系的构建. 采用夹板连接、模板PCR、Tail PCR等方法成功在Oct4、Sox2、Klf4、cMyc和EGFP mRNA的ORF两端连接上特殊的5’UTR和3’UTR，NTP mix进一步进行3’端加帽修饰，采用MEGAscript T7 Kit试剂盒进行体外转录，获得了基因转染和iPS细胞诱导所需的修饰mRNA。分别采用Lipofectamine 2000和Lipofectamine RNAiMax脂质体将Oct4、Sox2、Klf4、cMyc和EGFP的修饰mRNA混合物（3:1:1:1:1）连续转染大黄鱼肝脏、脾脏和肾脏的原代细胞，EGFP的mRNA转染率均达到20%以上。并对转染后的细胞进行了连续培养以诱导iPS细胞的产生。. 3. 组织细胞的原代培养. 采用组织块贴壁培养法和离散细胞培养法进行大黄鱼肝脏、脾脏、肾脏等组织的原代细胞培养并成功传代，为iPS细胞诱导提供了足量优质的体细胞。. 4. iPS细胞的鉴别. 采用碱性磷酸酶染色法检测诱导后的细胞是否呈阳性表达； qRT-PCR技术检测Oct4、Sox2和Nanog的表达情况；Western Blot检测干细胞表面标志物Oct4、Sox2和Nanog的表达情况；还进行了染色体核型分析，以确定细胞是否出现遗传变异等。这些实验结果目前正在整理补充中，后续会以论文的形式发表提交。