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

虽然有报道称，将慢病毒载体基因组随机插入胚胎中可以产生转基因绒猴，但这种动物的表型会因整合位点而不同。为了建立更先进的方法，研究了腺相关病毒载体（rAAV）介导的到绒猴胚胎中的转导，以及将几种基因编辑系统包含到rAAV中。结果如下：1）由于添加表达AAV 2 Rep的rAAV 5，rAAV 2基因组整合到绒猴细胞系中的增强。2)携带具有phi-Gamma催化结构域的TALE的AAV载体质粒的构建。3)通过转染携带CRISPR/Cas9系统的AAV载体质粒诱导HEK 293细胞的indel突变，并从质粒产生rAAV。4)rAAV 9介导的向绒猴胚胎的有效转导，以及随后在ICM中的稳健转基因表达。这些结果和发现将有助于生产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.