The presented methodology of embryonic chicken lens microinjection of a RCAS retrovirus is meant as a tool for studying in situ function and expression of proteins during lens development. Compared to other approaches, such as transgenic models and ex vivo cultures, the RCAS replication competent avian retrovirus provides a highly effective, rapid, and customizable system for expressing exogenous proteins in chick embryos. Targeted gene transfer can be confined to proliferative lens fiber cells without the need for promoters.
There is prominent potential for utilizing this methodology to study lens development, differentiation, cellular communication, and disease progression, and further discovery and testing of therapeutic targets for lens pathological conditions such as cataracts. To begin, attach the borosilicate glass capillary to the rubber cushioned clips in the manual vertical micropipette puller. Set the heat temperature of the micropipette puller to 950 degrees Celsius and perform pre-pulling to obtain a thinner and softer glass capillary.
Then set the heat temperature to 790 degrees Celsius and perform a secondary pulling to produce final micropipettes. Using a micropipette grinder, carefully sharpen the tip opening to an outer diameter of 11 micrometers. Then place the micropipettes in a sponge clamping pad inside of a glass jar.
Incubate the fertilized chicken eggs until development stage 18 in a 37 degree Celsius humidified rocking incubator. Wipe down the working area thoroughly with 70%ethanol. After development, remove the eggs from the incubator.
Spray the eggs with 70%ethanol and allow them to air dry. Then place the egg on an egg holder with the larger end facing upwards. Using a pair of sharp toothed forceps, carefully tap on the larger end of the egg shell to create a two centimeter diameter hole.
Then remove the fragments of the eggshell. Place the egg on a dissecting microscope stage and locate the embryo. Next, use the fine dissecting forceps and scissors to cut off the amniotic membrane covering the top of the embryo.
Then place a 60 millimeter Petri dish over the opening of the egg. To begin, thaw the RCAS(A)retrovirus stock on ice. To avoid clogging the micropipette, centrifuge the solution at 10, 000 G for 10 seconds at four degrees Celsius and transfer the supernatant into a new tube while keeping the solutions on ice.
Place a stretched laboratory parafilm on a 35 by 10 millimeter cell culture dish and add one microliter of sterile PBS to the parafilm. Connect a prepared glass micropipette to an automatic picoinjector. Press the Fill Mode to fill the PBS into a micropipette, and then press the Inject Mode to test the allocated one microliter of liquid.
Place a second piece of stretched laboratory parafilm on a new cell culture dish and add one microliter of Fast Green-stained viral stock to the film. Lower the tip of the micropipette into the viral stock, and press the Fill Mode to fill the micropipette with one microliter of viral stock. Under a dissecting microscope, lower the filled micropipette connected to an automatic injector into the target region of the chicken embryo lens.
Then adjust the light source to provide a clear view of the lens vesicle. After ensuring the micropipette is within the correct location inside the lens lumen, inject five to 40 nanoliters of the viral stock. After injection, wait 45 seconds before gently removing the micropipette.
Next, check the successful microinjection by examining the Fast Green dye in the empty lumen of the lens without any leakage. Following examination, seal the eggshell opening with scotch tape and place the eggs in a 37 degree Celsius humidified static incubator. This study demonstrates the histological evaluation of the chimeric connexin 50 and 43 loops through immunofluorescence.
Using anti-flag labeling, exogenous connexins were distinguished from endogenous ones. The study also evaluates the interaction between the intracellular loop domain of connexin 50 and aquaporin zero.
