Xenopus laevis as a Model to Identify Translation Impairment

The overall goal of this procedure is to evaluate the first consequences of mutations in translation initiation factors without interference of newly transcribed mRNA or of transfection efficiency problems often occurring with eukaryotic cell studies. This is accomplished by first synthesizing mutant wild type and control, or GFP mRNA from corresponding plasmids. Next synthesized RNA is micro injected in XUS Lavis cytes at stage six, and their maturation is stimulated with progesterone.

Then the cyte maturation is evaluated by germinal vesicle breakdown visualization and some of the components of the MA kinase cascade whose activation depends upon moss protein expression are analyzed by western blot. Finally, mRNA Polyadenylation of moss and other mRNAs necessary for XPO cytes myosis recovery are studied. Ultimately kinetic cyte maturation.

Western blot and polyadenylation analyses are used to show a potential impact of protein expression when a translation initiation factor is mutated. The main advantage of this technique over our existing methods or reconstituted cellular system extracted from equally with germ or rabbit reticulocyte, is that the effects of a mutation introduced in an MR.NA will be quickly observable and easily studied in several xop levies. Cytes More generally.

This model has the advantages of simplicity with its single giant cells, and its use leads to a considerable amount of material for biochemical experiments. This process is also time efficient compared to stable cell line generation. This miso can help answer key questions in translation studies such as to better understand the role of specific domains of ation factors or to study Splicing of this factors.

We first add the idea for this method when we wanted to study the impact of a mutated translation initiation factor on protein synthesis without interference of transcription. By this way, we avoid potential feedback loops between these two ome mechanisms. Indeed, material transcript are stored and put.

Translation can be triggered with progesterone After defecating, xus lavos cytes and preparing CRN according to the text protocol, use 10 x mops and 6.6%formaldehyde to cast a 1.5%AROS gel. Prepare samples in a 0.2 milliliter tube with 8.8%formaldehyde, 60 percentfor AMIDE 0.1 volumes of 10 x mops and one microliter of CRNA incubate at 70 degrees Celsius for three minutes and put the tubes on ice for 10 minutes. Centrifuge the samples at 5, 000 times G for a few seconds before adding two microliters of gel loading buffer.

Run the samples at 90 volts for 20 minutes to disdain the gel. Soak it in nuclease free water overnight. Then analyze it to check the quality of the CNA.

Use a spectrophotometer to determine the CNA concentration and store the samples at minus 80 degrees Celsius to carry out micro injection of RNA. Use ND 96 medium to fill a scraped Petri dish one to two hours after defoliation. Arrange the cytes along a scraped lane in the dish.

Next, using a capillary micro pipette with a broken tip placed at a 45 degree angle. Insert the capillary tip about 150 to 200 microns deep into the equatorial zone below the pigmented animal area. Inject 30 nanograms of CRNA in 60 nanoliters into the first oocyte.

Then wait five to 10 seconds before removing the capillary tip to prevent the sample from leaking out. To inject the next cyte Manually move the dish. Transfer the injected cytes into 24 well culture plates filled with three milliliters of ND 96 medium and lead them at 19 degrees Celsius to trigger mitotic maturation.

Incubate the oocytes in ND 96 with two micrograms per milliliter of progesterone or PG at 19 degrees Celsius for 15 hours. Test the translation effect of wild type EIF 4G one CRNAs on the mutant phenotype according to the text protocol. To determine the extent of germinal vesicle breakdown under a stereo microscope.

Count the number of mature cytes after PG stimulation by the presence of a white spot at the black animal pole. Repeat the counting every hour until the 24th hour to carry out western blood analysis. Use the back and forth motions of a micro pipette tip at four degrees Celsius to homogenize 10 cytes in 200 microliters of the buffer shown here.

Centrifuge the samples at 10, 000 times G for 15 minutes to extract the cytoplasmic fraction in the middle. Phase the upper fraction corresponding lipids and the lower to cellular debris. Collect the cytoplasmic fraction and store an aliquot to determine protein concentration.

Combine the remaining at a one-to-one ratio with lamely or Biss sample buffer before running SDS page and western blotting. According to the text protocol. To carry out a poly ventilation assay, place five cytes per condition into 1.5 milliliter micro fuge tubes with one milliliter of nuclease free one XPBS wash the cytes.

Then under the fume hood, use an RNA extraction kit according to the manufacturer's instructions to isolate the RNA. After checking the integrity of the RNA and determining the concentration, prepare two ligation mixtures per CRNA condition with the following reagents to the first mixture. Add RNA from cytes non stimulated with pg and to the second mixture.

Add RNA from PG stimulated oocytes. Incubate at 37 degrees Celsius for one hour, and then at 65 degrees Celsius for 20 minutes. To inactivate the enzyme using a CD NA reverse transcription kit.

For each sample, prepare the RT mixture listed here. Add 10 microliters of the previously prepared ligation reaction and perform RT according to the manufacturer's instructions after carrying out PCR, using the following reaction mixture and conditions to each reaction at four microliters of loading buffer and run 10 microliters of each sample on a 3%agros gel at 110 bolts. Finally analyze the gel after 10 and 20 minutes to observe a change in size reflecting the length of the polyA tail and thus the RNA maturation, which is a prerequisite to their translation.

As shown here, the kinetic maturation of oocytes micro injected under control conditions are similar to wild type with similar numbers undergoing GVBD at 12 and 24 hours after PG stimulation, 24 hours after PG stimulation. The percent of oocytes reaching maturation is similar for wild type as well as water and GFP injected controls indicating that micro injecting with water or control or EIF 4G one CRNAs had little effect on oocyte myosis microinjection with the dominant negative EIF 4G one, however, resulted in a dramatic decrease in GVBD even after PG stimulation. This figure shows that the maturation defect can be restored the wild type CRNA as the ratio of wild type to dominant negative EIF 4G one CRNA increases.

So does the percent of cytes that undergo maturation as expected. No endogenous moss expression is detected in the absence of PG stimulation and overexpression of EIF 4G one dominant negative has little impact on endogenous moss in agreement with previous results. In addition, Aurora AEEG two, which acts upstream of moss induction, shows no evidence of protein deregulation or of its phosphorylated form induced after PG stimulation.

Conversely, irk two phosphorylation, which is induced by moss, is inhibited in the presence of the dominant negative EIF 4G one. Once master, this technique can be done in five days if it is performed properly. While attempting this procedure, it's important to remember to not exceed 120 nanoliters of CNA with a concentration below certain nanograms Following this procedure.

Although method like polysome profiling analysis can be performed in order to answer additional question like defining which RNA might be poorly or highly translated.