Article Text

  1. T. L. Lombardi,
  2. J. Chen
  1. Keck School of Medicine, Los Angeles


Purpose Photoreceptor cells rapidly lose their structure and function when they are cultured, thus requiring an in vivo setting for their studies. Genetic manipulation of photoreceptor cells using traditional transgenic mouse techniques is time consuming and costly. We explored the recently described method of in vivo electroporation as an alternate means to introduce foreign DNA constructs into mouse retinas. Ultimately, we will use this method for future studies of wildtype and mutant rhodopsin synthesis and turnover in transfected photoreceptor cells. Such studies may help to better understand the effects of some of the rhodopsin mutations that are causal for autosomal dominant retinitis pigmentosa.

Methods To test in vivo electroporation technique, a DNA plasmid containing GFP driven by the ubiquitin promoter was generated. Under a dissecting microscope, DNA was injected into the subretinal space of newborn CD1 mice using a Hamilton syringe. After injection, tweezer-type electrodes soaked in PBS were placed on the sides of heads of pups and used to create five 80-V pulses. Electroporated retinae were harvested 21 days after injection, fixed, and cut on a cryostat in sections of 20 μm. Cryosections were then examined with a Zeiss Axioplan fluorescent microscope. To examine turnover of mutant rhodopsin in photoreceptors, constructs were made that use the rhodopsin promoter to drive expression of chimeric proteins containing timer fluorescence (dsRed-E5) and either wildtype or mutant rhodopsin.

Results When viewed under the fluorescent microscope, cryosections of retinae containing the ubiquitin-GFP construct demonstrated expression of GFP in outer nuclear layer of photoreceptor cells as well as cells in the inner nuclear layer. Plasmids containing mouse rhodopsin promoter driving expression of wildtype rhodopsin-dsRed-E5 and rhodopsin mutant-dsRed-E5 were constructed and verified by sequencing.

Conclusions Expression of GFP in the retinae of P21 mice after DNA injection demonstrates in vivo electroporation is a possible means to study mutant rhodopsin in mice. Future experiments will use in vivo electroporation to study the turnover of the mutant rhodopsin-dsRed-E5 constructs.

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