Transuterine microinjection into the embryonic day 11.5 mouse otocyst (A) An aqueous solution (0.1%) of fast green dye was microinjected into the left otocyst through the uterine wall at E11.5. The embryo was dissected free of the uterus and the extraembryonic membranes and its left side was imaged. The otocyst is located superficially in the lateral head mesenchyme, dorsal and slightly medial to the main trunk of the primary head vein (black arrow) and approximately midway between its rostral and caudal branches (white arrowheads). The trunk of the primary head vein forms the base of an American football goalpost and its rostral and caudal branches form the uprights. The otocyst displays a clamshell-shaped vestibule and tapered dorsal endolymphatic duct. Abbreviations: e, left eye; 4^th, nascent 4^th ventricle; v, visceral yolk sac. (B) The same embryo imaged in panel A immediately after transuterine microinjection of dye into the left otocyst. The uterus and extraembryonic membranes are intact. The 4^th ventricle, eye, and rostral branch (white arrowhead) of the primary head vein (black arrow) are visible. The caudal branch of the primary head vein is not apparent. (C) The left side of the E11.5 mouse embryo prior to transuterine microinjection into the otocyst. The transillumination scheme in this image highlights the caudal branch (white arrowhead) of the primary head vein (black arrow), but the rostral branch is not clearly visible. Since the otocyst cannot be visually identified at E11.5, its location is inferred by interpretation of the anatomical landmarks provided by the primary head vein vasculature. Scale bar = 1 mm and applies to all panels.

Electroporation-mediated transfer of an expression plasmid encoding green fluorescent protein transfects progenitors that give rise to the organ of Corti. An expression plasmid encoding green fluorescent protein (GFP) driven by the human elongation factor 1-α promoter (EF1-α) was electroporated into the E11.5 mouse otocyst. The inner ear was harvested six days later at E17.5 and fixed in 4% paraformaldehyde in PBS for 12 hours. The cartilaginous otic capsule and the cochlear lateral wall were removed and the whole mount preparation was imaged in panel A. GFP expression is detectable in the base, midbase, and proximal apex of the cochlea. (B) Laser confocal microscopy of a representative 100 μm section of the EF1-α /GFP-transfected cochlea immuostained with an antibody against myosin VIIa (red) to identify the single row of inner hair cells and three rows of outer hair cells. Several supporting cells (sc), pillar cells (pillar), and outer hair cells (ohc) express GFP. These data indicate that progenitors giving rise to supporting cells and hair cells of the organ of Corti were transfected and expression of the transgene was maintained in differentiated cell types of the maturing cochlea. Scale bar in A = 100 μm and in B = 10 μm.

Fabrication of a transuterine microinjection pipette. A pipette was pulled with the P-97 micropipette puller using the Pressure:Heat:Pull:Velocity:Time settings are indicated in Subheading 3.3, step 1. (A) The outer diameter of the unpulled shaft is 1.5 mm and the length of the tapered part is ~12 mm. The tip of this pipette was imaged in 3 successive stages of preparation (B-D). (B) The approximate location of the manual break point at the tip of the pipette is indicated by the arrowhead. The pipette tip was broken by pinching the glass with biologie #5 forceps (Fine Science Tools). (C) The broken pipette presents a bifurcated, coarse tip with particulate debris on the outside of the glass. (D) The pipette tip was beveled with the BV-10 Beveler to replace the jagged leading edge and establish a 20 degree tip. The final outer diameter of the injection pipette is ~20 μm (target range is 18-24 μm). Scale bar = 50 μm (applies to B-D).

In vivo electroporation of the embryonic day 11.5 mouse otocyst. (A) The uterus was transilluminated with light from a fiber optic cable whose output end (fo) was directly in contact with the irrigated uterus. The left otocyst (o) of the E11.5 mouse embryo was injected with fast green solution and its gross morphology is discernable beneath the nascent 4^th ventricle (4^th) in the caudal hindbrain. The insulated surface of the cathode (c) and the reflective, platinum surface of the anode (a) were grossly positioned around the uterus to flank the embryo. (B) Gentle compression of the uterus with the tweezer-style electrodes forced a counter-clockwise rotation of the embryo, placing the injected otocyst toward the center of the 5 mm cathode-anode field. The goal is to drive negatively charged DNA into ventral progenitors within the otic vesicle. (C) Bubbles (b) on the surface of the uterus after execution of the 5-pulse train appear as graininess in the image. Light pressure from the thumb (t) reorients the embryo within the uterus to facilitate placement of the electrodes (panel A). Both the fiber optic light head and the thumb are removed from contact with the uterus prior to triggering the square wave pulse train. Scale bar = 1 mm and applies to all panels.