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Phenylketonuria (PKU) is a monogenic disorder resulting from Phenylalanine hydroxylase (PAH) enzyme deficiency in liver hepatocytes. Untreated patients have clinical signs including growth retardation, microcephaly, short stature and low IQ. As the production of tyrosine from Phenylalanine is generally dependent on this enzyme, mutant PAH will result in accumulation of byproducts (liver cells), that is generated during deficient phenylalanine metabolism. This study aimed to produce a liver cell model harboring deletion in PAH, which could be applied to investigate mutations and their effect on cell metabolism as well as response to drugs and supplement administration. A large deletion in exon 6 of PAH was created by CRISPR/Cas9 system, using pSpCas9(BB)-2A-Puro (PX459) V2.0 and pSpCas9(BB)-2A-GFP (PX458) vectors. HPLC method was used to measure the level of phenylalanine and tyrosine in the cell with deleted PAH gene. Contrary to our expectation, a decrease in the phenylalanine level was observed in the culture medium and inside the cell, but the tyrosine level exhibited an anticipated decrease in the culture medium and inside the cell. |
