Determination of the Apo E genotype using LightCycler Apo E mutation detection kit.

Apolipoprotein E (Apo E) is found to be genetically polymorphic. There are 3 common alleles designated as E2, E3 and E4. Polymorphism of the Apo E DNA is associated with the risk increase of many diseases such as dyslipidemia, cardiovascular diseases, Alzheimer's diseases, etc. Hence, the interest in genotyping of the Apo E is now rising for the purpose of pre-diagnosis. The aim of this study was to characterize the Apo E DNA polymorphism using LightCycleríApo E mutation detection kit (Roche) in the Thai normal healthy subjects. The genomic DNA was extracted from the blood of 133 normal healthy subjects using DNA extraction kit (Roche). Exon 4 of the Apo E gene was amplified by the extracted genomic DNA using the real-time PCR. The simultaneous analysis of the two polymorphic codons (Codons 112 and 158) in a single reaction was conducted by using the two reporter dyes with the different excitation and emission spectrum LightCycler-Red 640 (LC-Red 640) and LC-Red 705 followed by the color compensation software to correct the temperature-dependent crossover among the emission spectra of the dyes. The different genotypes were then determined by performing the melting curve analysis in the two different channels. The results showed that the allele frequencies of the Apo E2, E3 and E4 were 0.26, 0.63 and 0.11 respectively and the genotype frequencies of the E2/3, E2/4, E3/3, E3/4 and E4/4 were 47.37, 5.26, 32.33, 14.28 and 0.75% respectively. This study found that E2/3 was the most common genotype of the Apo E. In conclusion, the LightCycler (LC) allelic discrimination method to genotype the Apo E is rapid, simple, reliable and strongly recommended for being successful diagnostic tests in the future.

In vitro osteogenesis from human skin-derived precursor cells.

Embryonic tissue and organ development are initiated from three embryonic germ layers: ectoderm (skin and neuron), mesoderm (blood, bone, muscle, cartilage and fat) and endoderm (respiratory and digestive tract). In former times, it was believed that cell types in each germ layer are specific and do not cross from one to another throughout life. A new finding is that one tissue lineage can differentiate across to another tissue lineage, and this is termed transdifferentiation. We were interested in studying the transdifferentiation of skin-derived precursor cells (ectoderm layer) to osteoblastic cells (mesoderm layer). Human skin-derived precursor cells (hSKP) were isolated and induced into an osteoblastic lineage using osteogenic induction medium (alpha-MEM plus 10% fetal bovine serum supplemented with ascorbic acid, beta-glycerophosphate and dexamethasone). The specific characteristics of osteoblastic cells, including the expression of enzyme alkaline phosphatase, the deposition of mineral and the expression of osterix, bone sialoprotein and osteocalcin, were detected only from the inductive group. The results in our study show that SKP from human skin are a practically available source for osteogenesis. The samples are easily obtainable for autologous use with a high expansion capacity.