Abstract
We previously demonstrated that the injection of pregnant wild-type female mice (carrying enhanced green fluorescent protein (EGFP)-expressing transgenic fetuses) at embryonic day (E) 12.5 with an all-in-one plasmid conferring the expression of both Cas9 and guide RNA (targeted to the EGFP cDNA) complexed with the gene delivery reagent, resulted in some fetuses exhibiting reduced fluorescence in their hearts and gene insertion/deletion (indel) mutations. In this study, we examined whether the endogenous myosin heavy-chain α (MHCα) gene can be successfully genome-edited by this method in the absence of a gene delivery reagent with potential fetal toxicity. For this, we employed a hydrodynamics-based gene delivery (HGD) system with the aim of ensuring fetal gene delivery rates and biosafety. We also investigated which embryonic stages are suitable for the induction of genome editing in fetuses. Of the three pregnant females injected at E9.5, one had mutated fetuses: all examined fetuses carried exogenous plasmid DNA, and four of 10 (40%) exhibited mosaic indel mutations in MHCα. Gene delivery to fetuses at E12.5 and E15.5 did not cause mutations. Thus, the HGD-based transplacental delivery of a genome editing vector may be able to manipulate the fetal genomes of E9.5 fetuses.
Highlights
In murine fetuses at mid-gestational stages (from embryonic day (E) 9.5 to E12.5; the day the vaginal plugs are found is designated as E0), there are many organ anlages, and organogenesis occurs actively [1,2,3,4,5,6]
The method used for the DNA injection was based on hydrodynamics-based gene delivery (HGD) using pregnant B6C3F1 mice to target an endogenous gene (MHCα)
To identify which embryonic stages were amenable to HGD-based transplacental gene delivery (TPGD)-GEF, injections were performed on E9.5, E12.5, or E15.5
Summary
In murine fetuses at mid-gestational stages (from embryonic day (E) 9.5 to E12.5; the day the vaginal plugs are found is designated as E0), there are many organ anlages, and organogenesis occurs actively [1,2,3,4,5,6]. Gene delivery targeted to mid-gestational fetuses is largely divided into two routes: one is in utero gene delivery, based on an injection of NAs into fetuses exposed externally with subsequent electroporation at the injected site, and the other is transplacental delivery of NAs complexed with DNA delivery reagents into pregnant females [8]. Neural stem cells located at the basal layer of the zona ventricle of the fetal brain have been successfully transfected [9] The latter is based on systemic transfection of fetal cells by NAs delivered via the blood stream after passing through the transplacental barrier; in this case, the NAs are complexed with DNA delivery reagents and administered through a tail vein injection [10]
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