The overall goal of this amended cell culture protocol is to supply a rapid phenotypic assessment of allelic variance in Fabry and Pompe disease. This method can help to answer key questions in the field of lysosomal storage diseases, such as prognostic outcomes and therapeutic decisions. The main advantage of this technique is that it is highly reproducible and that it can aid in the development of novel drugs, such as pharmaco-chaperones.
To carry out site-directed mutagenesis, begin by using the reference sequences NM 00169.2 and NM 00152.4 as templates for the mutagenesis of GLA and GAA genes, respectively. Use the free Primer Design tool to support primer design. Then, have a set of high-purity, salt-free primers synthesized by a commercial provider, with sense and antisense primers carrying one of the respective sequence modifications, central to their length, to individually introduce the mutation.
Prepare the reaction mixture in a 50 microliter volume and run the PCR program using standard conditions provided by the manufacturer and as listed in the text protocol. Following PCR, add one microliter of Dpn1 restriction enzyme and continue to incubate the reaction vials at 37 degrees Celsius for one hour. After transforming the plasma DNA into competent E.coli cells and preparing plasmids of the desired transformance according to the text protocol, use a suitable molecular biology tool to analyze the sequence.
When the desired mutation is detected and no further sequence abnormality compared to NM 000169.2 for alpha-galactosidase A or NM 000152.4 for acid alpha-galactosidase is seen, select the clone for transfection-grade plasmid purification. Determine the purity of the DNA by measuring the absorbance in a spectrophotometer. Following the cultivation of HEK293H cells in a T75 culture flask according to the text protocol, 24 hours prior to the transfection, use PBS without calcium or magnesium to wash the cells once.
With 0.05%Trypsin-EDTA, harvest the cells and in the cavities of a 24-well culture plate use 500 microliters of DMEM supplemented with 10%FBS to seed 1.5 times 10 to the five cells. Carry out cell transfection according to the manufacturer's manual. Using a mixture of one microgram of plasmid DNA dissolved in 100 microliters of serum-free DMEM and 2.5 microliters of transfection reagent.
Incubate the solution for 20 minutes at room temperature, then add it to the cells in a dropwise manner. After a period of four hours at 37 degrees Celsius and 5%CO2, remove the medium containing the transfection reagent and add 500 microliters of fresh DMEM with 10%FBS and 1%penicillin-streptomycin. As an option during this step, add DGJ or DNJ to the culture medium where intended.
On the day of the harvest, remove the cells from the incubator. Then aspirate the medium and use PBS with calcium and magnesium to carefully wash the cells two times. This step is critical because DGJ and DNJ are inhibitors of both enzymes, and therefore any left over would invalidate the test.
Following the wash, add 200 microliters of deionized water directly on top of the cells. Then rinse the cells from the plate and transfer them to a 1.5 milliliter reaction tube. Place the samples in an appropriate foam rack and vortex them for five seconds to make the lysis more efficient.
Then alternate the samples between liquid nitrogen for 10 seconds and a room-temperature water bath until the thawing is complete. After repeating the homogenization procedure five times, spin the samples for five minutes at 10, 000g. Then transfer the supernatant into a new reaction tube.
To carry out the BCA assay, prepare a fresh tube for each sample containing 40 microliters of deionized H20 and add 10 microliters of sample. Mix each solution by briefly vortexing and transfer 10 microliters of each sample in triplicate into the cavities of a 96-well plate. To prepare a standard curve, dilute a 2 milligram per milliliter BSA stock solution and deionized H2O according to the text protocol.
After combining reagent A and reagent B, start the reaction by adding 200 microliters of the BCA reagent solution and incubate the samples in the dark at 37 degrees Celsius and 300 rpm on an orbital shaker for one hour. Then measure the absorbance at 560 nanometers in a plate reader. The samples typically contain between one and 1.5 micrograms of protein per microliter.
Dilute the calculated amount of each sample and pipette it into fresh 1.5 milliliter reaction tubes to obtain 0.05 micrograms of alpha-galactosidase A or 0.5 micrograms of acid alpha-glucosidase per microliter of solution. Vortex the samples for five seconds again and pipette 10 microliters of this dilution in duplicate into a 96-well plate. Start the reactions by adding 20 microliters of the respective substrate solution.
For alpha-galactosidase A, add two millimolar 4-methylumbelliferyl alpha-D galactopyranoside, or 4-MU-gal, in 0.06 molar phosphate-citrate buffer, pH 4.7. For acid alpha-glucosidase, add 2 millimolar 4-methylumbelliferyl alpha-D glucopyranoside, or 4-MU-glu, in 0.025 molar sodium acetate, pH 4.0. Incubate the enzyme reactions for one hour in the dark at 37 degrees Celsius and 300 rpm on an orbital shaker.
Then terminate the reaction by adding 200 microliters of 1.0 molar pH 10.5-adjusted glycine sodium-hydroxide buffer. Prepare a standard curve of 4-MU from a 0.01 milligram per milliliter stock according to the text protocol and pipette 10 microliters of the solutions in duplicate into a 96-well plate. Add 200 microliters of the 1.0 molar glycine sodium-hydroxide buffer to each well in order to adjust the volume and pH.
Finally, measure the enzyme activity in a florescence reader equipped with the appropriate filter set. And analyze the data using the appropriate software for the fluorescence-reader device. To assess the efficiency of GLA gene mutagenesis, the mutations were classified into three categories and revealed that about 66.5%were obtained in the first attempt.
A further 25%could be obtained after a slightly modified second PCR. In category three, more effort, such as primer redesign, had to be undertaken to yield the desired clone. This table refers to the enzyme-activity measurement results for three alpha-galactosidase A and three acid alpha-glucosidase mutations either left untreated or treated with DGJ and DNJ.
The data are displayed as absolute values for the substrate turnover and has relative values normalized to the wild-type enzyme. The absolute enzyme activity data was corrected for endogenous enzyme activity of the HEK293H cells using cells transfected with an empty pcDNA 3.1 vector. Enzyme activity values were normalized to wild-type enzyme from corresponding experiments, which explains the deviation of relative values between the different mutants.
Once mastered, the post-cell culture fraction of this experiment, which represents most of the hands-on time, can be done within four hours. After its development, this technique paved the way for researchers in the field of lysosomal storage diseases to explore genotype-phenotype correlations in Fabry and Pompe disease. This protocol can aid in the development of novel drugs in lysosomal storage diseases.
