Ultrasound-guided in utero injections allow for studies of eye development that were not feasible previously. This includes spatiotemporal control of both gain- and loss-of-function. The choice of gene delivery method, by means of virus or electroporation, depends upon the application and should be chosen according to the age of delivery, number of cells to be targeted, stability of expression, size of construct(s), and whether or not multiple constructs need to be delivered. Viral infections at E9.5–E10.5 usually resulted in large clones composed of multiple columns of densely packed retinal cells, which should enable studies of retinal physiology. Similarly, studies of degeneration should be possible, because by adjusting the viral titer, clones covering up to 20% of the retinal surface area can be achieved. Animals infected with pQCXIX could be easily identified by GFP signal through the lens and thus can be selected for follow-up studies, for example, for physiology. In addition to gene transfer into the retina, these methods also yielded gene transfer of the RPE and sclera. Gene transfer into other anterior chamber structures such as the lens, the cornea, and the ciliary margin should also be possible, facilitating studies of glaucoma, cataract, and corneal development and diseases.
Gain-of-function by viral infection can be performed by overexpression of a gene in a broad or cell type-specific manner by use of a cell type-specific promoter that drives expression of the gene of interest. Cell type-specific overexpression can also be achieved by infecting a Cre-expressing mouse line with a virus where a flox-stop cassette precedes the gene of interest. Similarly, a virus with a cell type-specific promoter that precedes a flox-stop cassette in front of the gene of interest injected into a Cre-line with a different promoter will result in overexpression at the intersection of two different promoters. Gain-of-function by electroporation is straight forward as multiple plasmids can be electroporated at the same time. In addition, spatiotemporal control without the use of Cre-lines has been reported successfully for electroporations (Matsuda and Cepko, 2007
). Analogous to gain-of-function, loss-of-function can be achieved by introducing a Cre or Flp by means of viral infection or electroporation into an animal engineered to delete a locus of interest. An alternative approach is to deliver RNAi, by means of a retroviral vector or electroporation.
Ultrasound-guided in utero injections during mouse eye development provides a more rapid screening process for novel gene function and promoter activity than previously afforded by the use of genetically modified mice. The ability to perform experiments, including the generation of loss-of-function clones similar to those that have advanced the understanding of Drosophila
eye development, will enable scientists to dissect the cellular interactions that govern eye development in mouse. Screening for disease models generated by RNAi- or Cre-mediated loss-of-function, as well as rescue experiments in retinal degeneration mutants, should be straightforward. In addition, it is likely that these methods can be adapted to other species, thereby expanding the production of disease models to organisms where germline engineering is impossible or impractical. While this work was in progress, Garcia-Frigola et al., reported successful electroporation in utero into the embryonic mouse eye (Garcia-Frigola et al., 2007
). However, they did not use ultrasound-guided injections and expression was confined mainly to amacrine and ganglion cells. Due to the lack of ultrasound-guided assistance, delivery of genes earlier than E13 is also not feasible by their method as the developing eye is too small to be targeted.