UV Irradiation: A Method to Integrate Extrachromosomal Arrays
Transcrição
– Creating extrachromosomal DNA arrays by microinjection– the simplest method para generate transgenic worm strains– results de genetic elements that are not stably transmitted during cell division and, therefore, exhibit variable inheritance rates and expression levels. The goal of integrating the array into a chromosome is para lessen this genetic instability and variability.
To do so, start with an age-synchronized population of L4 transgenic worms, which carry an extrachromosomal array previously introduced by microinjection and allow them para grow into gravid adults. Transfer these adults para new plates and allow them para lay eggs until there are at least 30 eggs on each plate. Remove the adults and allow the eggs para grow up until they reach the L4 stage. Then, irradiate the worms with UV light. This causes random double-strand breaks de the genomic DNA where the worms DNA repair machinery may arbitrarily incorporate the array.
After an overnight recovery period, count the number of surviving worms. A small percentage of dead worms indicates efficient irradiation. Observe subsequent generations para isolate homozygous integrated transgenic lines. In the example protocol, we will integrate a high transmission rate extrachromosomal array carrying a fluorescent co-injection marker expressed de the pharynx.
– Evaluate the transmission rate of the transgenic line para be integrated. For each transgenic line, pick 10 fluorescent gravid adults onto 10 separate culture plates under a fluorescent stereoscope. Culture the animals de a worm incubator set at a temperature permissive para reproduction of the genetic background.
Monitor the progeny each day para evaluate which developmental stage the co-injection marker is expressed and can be best observed. Using fluorescent stereoscopy, evaluate the transmission rate of the progeny by determining the percentage of fluorescent progeny. For the transgene integration, select the transgenic line with the highest transmission rate.
Obtain a population of transgenic animals synchronized at the L4 larval stage for integration. Pick 30 fluorescent gravid adults onto five culture plates. After the worms have laid eggs for three para four hours at 15 degrees Celsius, check for the presence of at least 30 eggs by plate, and then, eliminate the adults from the plates.
Culture the animals de a worm incubator until the progeny reach the L4 larval stage. Place each plate containing 15 para 20 fluorescent transgenic L4 animals de a UV crosslinker with the lids removed. Irradiate the worms.
For recovery, incubate the irradiated worms overnight at 15 degrees Celsius. Check for the number of animals that are alive. In our hands, a survival rate of around 80% para 90% is adaptive for an efficient irradiation. Grow the irradiated animals at 15 para 25 degrees Celsius until the progeny have reached the developmental stage allowing for the observation of the co-injection marker expression.
