In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

Summary

This study describes the development of an in vitro electroporation technique that allows for the manipulation of gene expression in the lower rhombic lip of midgestation embryos.

Abstract

The rhombic lip is an embryonic neuroepithelium located in the hindbrain at the junction between the neural tube and the roofplate of the fourth ventricle (reviewed in ¹). The rhombic lip can be subdivided into the upper rhombic lip (URL) which encompasses rhombomere 1 (r1) and generates neurons of the cerebellum and the lower rhombic lip (LRL) which gives rise to diverse neuronal brainstem lineages ^2-4. LRL derivatives include the auditory neurons of the cochlear nuclei and those of the precerebellar nuclei that are involved in regulating balance and motor control ^5-8. Neurogenesis from the LRL occurs over a large temporal window that encompasses embryonic days (E) 9.5-16.5^{5, 9}. Different neuronal lineages emerge from the LRL as postmitotic cells (or are born) during distinct developmental days during this neurogenic window.

Electroporation of gene expression constructs can be used to manipulate gene expression in LRL progenitors and can potentially change the fate of the neurons produced from this region ^10-12. Altering gene expression of LRL progenitors in the mouse via in utero electroporation has been highly successful for manipulating lineages born on embryonic day E12.5 or later ^{10, 12-14}. In utero electroporations prior to E12.5 have been unsuccessful primarily due to the lethality associated with puncturing the fourth ventricle roofplate, a necessary step in delivering exogenous DNA that is electroporated into the LRL. However, many LRL derived lineages arise from the LRL earlier than E12.5 ⁹. These earlier born lineages include the neurons that comprise the lateral reticular, external cuneate, and inferior olivary nuclei of the precerebellar system which function to connect inputs from the spinal cord and cortex to the cerebellum ⁵. In order to manipulate expression in the LRL of embryos younger than E12.5, we developed an in vitro system in which embryos are placed into culture following electroporation.

This study presents an efficient and effective method for manipulating the gene expression of LRL progenitors at E11.5. Embryos electroporated with green fluorescent protein (GFP) driven from the broadly active CAG promoter reproducibly expressed GFP after 24 hours of culture. A critical aspect of this assay is that gene expression is only altered because of the expression of the exogenous gene and not because of secondary effects that result from the electroporation and culturing techniques. It was determined that the endogenous gene expression patterns remain undisturbed in electroporated and cultured embryos. This assay can be utilized to alter the fate of cells emerging from the LRL of embryos younger than E12.5 through the introduction of plasmids for overexpression or knock down (through RNAi) of different pro-neural transcription factors.

Protocol

1. Preparations Prior to Electroporation

1. Amplify the DNA for electroporation by a maxi prep (Prime-It or Qiagen). The concentration of the DNA should be a minimum of 1 mg/mL for efficient uptake.
2. Remove 495 μL of DNA and mix with 5 μl of 0.01% Fast Green in 1 X PBS (phosphate buffered saline) in a microcentrifuge tube.

2. Embryonic Harvest

1. Establish timed matings of CD-1 mice (Harlan). Check for the presence of vaginal plugs and regard the date a vaginal plug is observed as embryonic day (E) 0.5. Embryos will be harvested 11 days after visualizing plug (E11.5).
2. Place sterili....

Discussion

The in vitro electroporation technique presented in this study is a novel methodology that can be efficiently utilized to manipulate gene expression in embryos younger than 12 days of gestation. Placement of the embryos into culture permits expression of the introduced gene and circumvents the lethality observed when electroporated embryos are allowed to remain in vivo. This technique allows for the manipulation of gene expression in embryonic progenitors that were previously inaccessible for e.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cryostat	Leica Microsystems	CM-1850
Biologie tip Dumoxel treated DUMONT forceps	Fine Science Tools	11252-30
20 mm MORIA perforated spoon	Fine Science Tools	10370-17
ECM 830 Square Wave Electroporation Generator	BTX (VWR)	47745-928
Harvard Apparatus 7 mm Tweezertrodes* Electrodes	BTX (Fisher)	BTX450165
Fisher Isotemp CO2 Incubator	Fisher Scientific	1325525
NAPCO CO2 Gas Regulator	Fisher Scientific	15497020
12 Well Tissue Culture Plates	BD Falcon (Fisher)	877229
HyClone Liquid Media DMEM/F-12 (1:1); With L-Glutamine and HEPES; 500mL	Thermo Scientific (Fisher)	SH3002301
HyClone* Donor Equine Serum	Thermo Scientific (Fisher)	SH3007402
Fetal Bovine Serum, Qualified, Heat Inactivated	Invitrogen	16140-063
cellgro* 10,000 IU Penicillin, 10,000μg/mL Streptomycin	Mediatech (Fisher)	MT-30-002-CI
HyClone* L-Glutamine L-Glutamine; 200mM in 0.85% NaCl	Thermo Scientific (Fisher)	SH3003401
Fast-Green	Fisher Scientific	AC41053-0250	0.01%