摘要
Oocytes,the irreplaceable gametes for generating a new organism,are matured in the ovary of living female animals.It is unknown whether any genetic manipulations can be applied to immature oocytes inside the living ovaries.As a proof-of-concept, we here demonstrate genetic amendments of zebrafish immature oocytes within the ovary.Oocyte microinjection in situ (OMIS) stimulates tissue repair responses,but some of the microinjected immature oocytes are matured,ovulated and fertilizable.By OMIS-mediated Cas9 approach,ntla and gata5 loci of oocytes arrested at prophase I of meiosis are successfully edited before fertilization.Through OMIS,high efficiency of biaUeUc mutations in single or multiple loci using Cas9/gRNAs allows immediate manifestation of mutant phenotypes in Fo embryos and multiple transgenes can co-express the reporters in Fo embryos with patterns similar to germline transgenic embryos.Furthermore,maternal knockdown of dnmt1 by antisense morphoino via OMIS results in a dramatic decrease of global DNA methylation level at the dome stage and causes embryonic lethalityprior to segmentation period.Therefore,OMIS opens a door to efficiently modify the genome and provides a possibility to re repair genetically abnormal oocytes in situ.
基金
the National Natural Science Foundation of China (31330052 and 31590832).
