Fetuses of time-dated pregnant Balb/c dams were injected at E16 as previously described (75). Briefly, under isoflurane anesthesia, a midline laparotomy was performed to expose the uterine horns. A dissecting microscope was used to identify the vitelline vein of each fetus, and a total volume of 5 μl (38.4 ng/μl mRNA) of the NP formulation was injected using an 80-μm beveled glass micropipette and an automated microinjector (Narishige IM-400 Electric Microinjector, Narishige International USA Inc., Amityville, NY). After successful injection, confirmed by visualizing clearance of the blood in the vein by the injectate, the uterus was returned to the peritoneal cavity and the abdomen was closed with a single layer of absorbable 4-0 polyglactin 910 suture.

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