The overall goal of this procedure is to integrate transgenic extra chromosomal arrays into serratus elegance genome using ultraviolet irradiation. First, select the transgenic line with the highest transmission rate possible, ideally greater than 80%Then UV irradiate the selected transgenic L four larvae single out four of the fluorescent transgenic F two animals from each selected F1 plate. Next screen, the F two plates for 100%fluorescent transgenic.
F three worms. Ultimately picking a fluorescent animals of the F three generation can show the 100%inheritance of the transgene. This experimental approach is well suited for transgenic lines that transmit extra chromosome arrays at a high rate.
Demonstrating the procedure will be Stephanie Belman, a technician from our laboratory. Start with 500 large Petri dishes containing nematode growth. Medium Seed each plate with 0.3 milliliters of saturated isia coli.
OP 50 culture. Evaluate the transmission rate of the transgenic line to be integrated for each transgenic line. Pick 10 fluorescent gravity adults onto 10 separate culture plates under a fluorescent stereoscope culture.
The animals in a worm incubator set at a temperature permissive to reproduction of the genetic background. Monitor the progeny each day to evaluate which developmental stage the co injection marker is expressed and can be best observed using fluorescent oscopy. 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 L four larval stage. For integration, pick 30 fluorescent GRA adults onto five culture plates.
After the worms have laid eggs for three to 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 in a worm incubator until the progeny reach the L four larval stage. Place each plate containing 15 to 20 fluorescent transgenic L four animals in 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 to 90%is adapted for an efficient irradiation. Grow the irradiated animals at 15 to 25 degrees Celsius until the progeny have reached the developmental stage, allowing for the observation of the co injection marker expression.
Transfer single fluorescent F1 animals onto separate culture plates. Keep these F1 plates at 15 to 25 degrees Celsius until the progeny reach an appropriate stage for the observation of fluorescent F two animals depending on the co marker used. In this particular case, we observed the worms as adults discard all F1 plates exhibiting either one, no progeny, indicating that the F1 animal was sterile or died, or two no, or only a few fluorescent F two animals indicating that the F1 animal did not transmit the transgene at the expected rate.
Single out four fluorescent transgenic F two animals from each selected F1 plate. When using fluorescent transgenes, pick F two worms with a high level of fluorescence as this could indicate that these animals are homozygous for the integrated array. Note that when picking F two animals, it is critical not to carry along eggs or larvae in order to avoid false negatives In the next step.
After growing the F two animals screen the F two plates for 100%fluorescent transgenic F three worms. The screen is quite rapid as the presence of a single non fluorescent worm indicates that the plate should be thrown away, single out eight fluorescent F three animals from selected plates to confirm the 100%inheritance of the transgene. If possible, keep several independent integrated transgenic strains.
Integrations of transgenes in two different lines, transmitting extra chromosomal arrays at about the same frequency were performed. Using the standard and the improved protocols, the time and the number of plates necessary per integrated line are optimized in the improved protocol. Next, we tested the hypothesis that a higher transgene transmission rate in a non-integrated line renders easier the integration of the transgene into the genome.
One particular transgene was integrated in lines transmitting at different rates using the improved protocol for the highest transmitting line. Three integrated lines were recovered from only 90 selected F1 animals while for the transgenic line transmitting at 50 to 60%One integrated line was recovered from 110 selected F1 animals. Following this procedure, different methods can be performed in order to answer additional questions like determining gene expression, pattern and regulation, protein localization and protein overexpression, or development of subcellular markers.
