CRISPR editing in mice is a work in progress. Zygote electroporation might make the work go faster.

In recent months, much has been written on the ways in which researchers are applying the genome editing technique CRISPR-Cas9 to alter the genome. The technology is being used to knock out or modify DNA in research mice in order to study disease phenotypes and develop new treatments.

There are two traditional methods of generating genetically engineered mice (GEM). The first generates knock-out and knock-in mice by gene targeting embryonic stem cells and then injecting targeted homologous recombinant embryonic stem cell clones into the mouse blastocyst. The second is the generation of transgenic mice by microinjection of nucleic acid into the fertilized mouse embryo. Both methods are time-consuming.

The clustered regularly interspaced short palindromic repeat or CRISPR-Cas9 genome editing system allows for more efficient generation of mice with a range of genetic alterations. The materials are injected into fertilized eggs to generate GEM animals in one simple step. But the microinjection process is technically demanding, labor-intensive and costly. Electroporation (EP) can help overcome these hurdles.

Zygote electroporation technology is a high throughput way to generate genetically modified animals. Electroporation induces temporary pores in the membranes of the fertilized embryos, which enables the genetic material to get inside the embryo more readily. Two thin platinum plates administer electrical pulses milliseconds in length.

A number of different research groups, including scientists from University of California-Berkeley, The Jackson Laboratory, and RIKEN BioResource Center in Japan, have been experimenting using electroporation technology.

In November, Shinya Ayabe, Special Fixed Term Contract Research Scientist at RIKEN BioResource Center demonstrated at Charles River an electroporation method for mouse zygotes developed by Dr. Ayabe. Also participating in the demonstration was Tomohiro Tamari, Group Leader of GEMS Embryology in Charles River Laboratories-Japan. (Charles River scientists were ultimately able to get genome-edited mice efficiently using this technology).

Below are pictures, taken during the demonstration, showing some of the key steps in using EP to deliver the CRISPR-Cas9 system into mouse zygotes. All photos were taken by Matt Newby, IT Principal Specialist in Web Development at Charles River, and an accomplished photographer.

 

 

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Photos 1-3 and 5-8 were shot with a Canon 70D, Canon EF 28-135 f/3.5-5.6 IS lens, and Canon 580EX II flash with a Fotove Ring Flash modifier to even out shadows on the subjects. A hotshoe-mounted flash with a modifier, or bounced off a wall or ceiling provides more even and directional light than relying on the ambient light in the room, without the harsh shadows the popup flash tends to make. Having the ring light modifier on the camera instead of off-camera flashes keeps the shooting “footprint” relatively small in a crowded lab.

Photos 4 and 9 were shot using an iPhone 6+ back camera, using an eyepiece projection technique through the eyepiece lens.