To assess the impact of virus storage on NEPTUNE efficacy, we stored the lentivirus for 3 months at −80☌. However, higher viral titers had a negative impact on embryo survival ( Figure S1F). Different viral titers resulted in dose-dependent transduction of brain between 5.7% and 99.7%, quantified by flow cytometry ( Figures 1H: 1 × 10 9 infectious units per milliliter, mean = 11.4% ± 5.1% SD 5 × 10 9ifu/mL, mean = 45.4% ± 0.5% SD 1 × 10 10 ifu/mL, mean = 63.8% ± 3.4% SD 2 × 10 10 ifu/mL, mean = 94.8% ± 6.3% SD). Flow-cytometry and CellProfiler quantification yielded highly correlated quantification of GFP + cells (r 2 = 0.9945, p < 0.0001, Figure S1E) CellProfiler typically identified 80% of GFP + cells compared with flow cytometry. This approach achieved widespread positivity, concentrated in the brain/head region at E13.5 ( Figure 1F), which was stable for at least 6 months ( Figure 1G). To optimize selection of females for surgery and take into account the 3Rs (replacement, reduction, refinement), ultrasound confirmation of pregnancy before surgery can be used for staging to ensure ideal conditions ( Figure S1D). This late neural plate stage, characterized by an elongated allantoic bud, precedes the appearance of the cranial neural (head) folds (HF), which arise at TS 11c ( Figure 1E). We compared appearance on ultrasound with the Theiler stage (TS) of embryos dissected out immediately after imaging and identified TS 11b as the optimal injection stage for 207 nL. A volume of 345 nL consistently resulted in resorption (325%–1,227% volume increase, mean = 615% ± 421% SD) and was excluded from further experiments. We therefore selected 207 nL (62%–162% volume increase, mean = 95% ± 38% SD) and 69 nL (9%–44% volume increase, mean = 28% ± 14% SD) for further study. Therefore, the best predictor of survival was not volume but the relative increase in amniotic volume, whereby an increase >90% resulted in embryo resorption ( Figures 1C and 1D). At E7.5, embryonic development and amniotic cavity size varies even within litters. Therefore, we drew the cutoff for acceptable survival rates at 70% and identified 207 nL as the highest permissible volume for normal survival ( Figure 1B). Sham surgery identified a baseline resorption rate of 0%–25%. To determine the optimal parameters for embryo survival and maximum transduction, we injected a range of volumes of viral resuspension buffer into the amniotic cavity (23–483 nL) at E7.5 and recorded the percentage of injected embryos per litter surviving at E13.5 ( Figures 1B and S1C). We therefore focused on E7.5 for in utero transduction, before neural fold fusion, when the amniotic cavity is discernible by ultrasound and the microcapillary needle tip can be accommodated in the cavity ( Figures S1A and S1B). Injections at E8.5 were too late to achieve consistently high levels of transduction, presumably because neural fold fusion is ongoing ( Figure S2). Neurulation stages can be discerned by ultrasound until neural fold fusion of brain at E9.5 ( Figure S1A).
Prior to neural tube closure in mice, around E7.5, the neural plate is exposed to the amniotic fluid and should be accessible to injected virus, allowing targeting of the future nervous system ( Figure 1A). NEPTUNE thus offers a rapid and cost-effective technique to test gene function in the nervous system and can reveal phenotypes incompatible with life. Sptbn2 knockdown induced dose-dependent defects in the neural tube, embryonic turning, and abdominal wall closure, previously unreported functions for Sptbn2. We used NEPTUNE to investigate Sptbn2, mutations in which cause spinocerebellar ataxia type 5. Knockdown of Olig2 by using NEPTUNE recapitulated the phenotype of Olig2 −/− embryos. Stable integration in >95% of cells in the brain enabled long-term overexpression, and conditional expression was achieved by using cell-type-specific MiniPromoters. To address this problem for the nervous system, we developed NEPTUNE (NEural Plate Targeting by in Utero NanoinjEction) to rapidly and flexibly transduce the neural plate with virus prior to neurulation, and thus manipulate the future nervous system. Genetic loss and gain of function in mice have typically been studied by using knockout or knockin mice that take months to years to generate.
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