Development of rAAV-mediated method to modify common marmoset pre-implantation stage embryo genome

虽然有报道将慢病毒载体基因组随机插入胚胎中产生转基因绒猴，但这种动物的表型会因整合部位的不同而不同。为了建立更先进的方法，对腺相关病毒载体(RAAV)介导的绒猴胚胎转导以及在rAAV中包含几种基因编辑系统进行了研究。结果如下：1)表达AAV2Rep的rAAV5促进了rAAV2基因组整合到绒猴细胞系中。2)含Phi-Gamma催化区的AAV载体载体的构建。3)用携带CRISPR/Cas9系统的AAV载体质粒对HEK293细胞诱导INDELL突变，并产生rAAV。4)rAAV9基因高效导入绒猴胚胎，并在ICM中高效表达。这些结果将有助于生产经基因组编辑的非人类灵长类动物。

Although generation of transgenic marmosets with random insertion of lentivirus vector genome into embryo was reported, the phenotypes of such an animal would be diversified depending on the integration site. To establish more advanced method, adeno-associated virus vector (rAAV)-mediated transduction into marmoset embryo, and inclusion of several gene-editing systems into rAAV were examined. The results were as follows: 1) Enhancement of rAAV2 genome integration into a marmoset cell line due to addition of rAAV5 expressing AAV2Rep. 2) Construction of AAV vector plasmid carrying TALE with phi-Gamma catalytic domain. 3) Induction of indel mutation to HEK293 cells by transfection of AAV vector plasmid carrying CRISPR/Cas9 system, and production of rAAV from the plasmid. 4) Efficient rAAV9-mediated transduction into marmoset embryo, and consequent robust transgene expression in ICM. These resulting objects and findings would be useful for production of genome-edited non-human primate.