This article describes a method for gene manipulation in genetically modified mouse pups using viral vectors. The procedure involves precise stereotaxic injections to achieve targeted expression of fluorescent proteins.
Take a genetically modified mouse pup carrying a loxP-flanked STOP cassette that blocks red fluorescent protein or RFP expression.
Secure the pup on a custom-made stereotaxic frame and cover it with ice to maintain anesthesia.
Disinfect the scalp and mark lambda as the reference point.
Align the injection needle to striatal coordinates relative to lambda and puncture the skull.
Retract the needle to the skull surface, then insert it into the target depth.
Inject the dye-mixed viral vector into the striatum at a controlled rate. The vector encodes green fluorescent proteins or GFPs and Cre recombinase.
Withdraw the needle slowly to prevent solution backflow.
Remove the pup and allow recovery.
In striatal cells, the virus releases its genome, enabling the expression of Cre recombinase and GFP. Cre excises the loxP-flanked STOP cassette, activating RFP expression.
Simultaneous GFP and RFP expression confirms successful gene manipulation.
Position the pup with the glove in the head tray and put some crushed ice around the latex sleeve to maintain the hypothermia anesthesia. Then scrub the pup's head with 70% ethanol and locate the lambda landmark. Mark it with a marker. Then aim the needle tip to the lambda and set the anterior, posterior, and medial lateral coordinates to zero.
Next, consulting a brain atlas, move the injection arm to the target site. For example, the striatum of P2 pups is 2.4 millimeters anterior to the lambda, 1.0 millimeters to either side of midline, and 1.7 millimeters ventral. Next, mark the position of fast green dye on the PE10 tube using a pen. Then slowly begin to penetrate the needle through the skin and skull until the needle tip contacts the surface of the skull.
Set the dorsal ventral coordinate as zero. Then lower the needle slowly to the target site. Once in position, wait for one minute to allow the parenchyma to return to its normal shape. Then run the microinjection program at 100 nanoliters per minute. During the injection, watch the fast green dye move in the PE tubing.
It is critical to slowly penetrate the needle through the skin and skull until the needle tip contacts the surface of the skull. During the injection, it is imperative to watch the Fast Green dye move in the PE tubing.
After the injection is completed, wait one minute and then slowly bring up the needle to one half of the penetrated DV depth. Then wait another 30 seconds before proceeding with the slow withdrawal of the needle from the pup's head.
After completion of the injection, it is important to wait one minute before slowly bringing up the needle to one half of the penetrated DV depth, then wait another 30 seconds before slowly withdrawing the needle from the pup's head.
After all the targeted sites have been injected, warm up the pup for 20 minutes in a 33 degree Celsius incubator.
Check on the pup every five minutes until it has regained sternal recumbency. Then return the pup to its dam.
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