Sanger sequencing which is a gold standard method for genotypic drug resistance testing has limited sensitivity in detecting HIV-1 drug resistance mutations (DRMs) at frequencies below 20% of viral quasispecies. Deep sequencing is an ultrasensitive method, which allows detecting such mutations and mutations detected by Sanger sequencing. A newly developed HIV-1 deep sequencing drug resistance assay has never been tested the accuracy with external quality assessment (EQA) program. The objective of this study was to test the accuracy of deep sequencing in detecting DRMs in HIV-1 protease (PR) and reverse transcriptase (RT) genes with five samples from ENVA 2015 HIV drug resistance typing EQA program. According to the 2015 EQA program report, deep sequencing could generate complete datasets of all five ENVA15 panel samples, which covered both PR and RT genes and comprised all IAS codons. In addition, deep sequencing detected 339 of 340 DRM codons and was awarded 99.86% of the overall sequence concordance. A manual reviewed bam file of ENVA15-08 sample was performed to investigate the incorrect codon and found at position 2,381 (PR-43) had a mixture of A (wild-type) at 83% and G (mutant type) at 16% which was identical to the expected results (R). Therefore, we reanalyzed the sequences of all samples by another pipeline found deep sequencing detected 334 DRM codons identical to expected results which comprised the incorrect codon and 333 DRM codons. In addition, the other 6 codons which comprised mutants at frequencies below 20% of viral quasispecies were partial concordance with the expected results because 2015 ENVA consensus sequences were created by aligning sequences summited by all participants whose almost all datasets were based on Sanger sequencing technology. In conclusion, deep sequencing has accuracy in detecting HIV-1 DRMs and would be adopted as a clinical laboratory routine in the near future.
Thalassemia is considered as a major health burden in the Southeast Asian and Indian populations. Patients with β-thalassemia display disease heterogeneity ranging from nearly asymptomatic to severe anemia with complications. Increased levels of fetal hemoglobin (HbF) can ameliorate the clinical severity of β-thalassemia patients. The HbF production is influenced by many quantitative trait loci (QTL). Three major HbF-QTLs are the BCL11A gene, the HBS1L-MYB intergenic region (HMIR), and the β-globin gene (HBB) cluster. Therefore, this study aimed to evaluate the influence of these genetic modifiers (BCL11A, rs766432; HMIR, rs4895411; and HBB cluster, rs2071348) on the HbF levels in Bangladeshi patients with β-thalassemia/HbE disease. The cohort study comprised of 90 patients with β-thalassemia/HbE disease, HbF levels ranged from 7.9% to 59.1%, from in and around the area of Chittagong, Southeast Bangladesh. Our results showed that levels of HbF were primarily influenced by alleles of the HBB cluster (rs2071348), and to a lesser extent by rs766432 HBL11A gene and HMIP (rs4895441) loci. The SNPs rs2071348 explained 12.5% of the variation in HbF levels, while 3.6% and 3.9% of trait variation were explained by rs766432 and rs4895441, respectively. In a case-control model of low and high HbF analysis, we found genotypes AC and AA (p = 2.0×10-4), the allele C (p = 2.0×10-4) for 2071348; genotypes AG and GG (p = 0.02), the allele G (p = 0.05) for rs4895441; all had a significantly higher frequency in the high HbF group than in the low HbF group, but rs766432 did not exhibit such features. Our results suggest these three major HbF-QTLs as the influencing phenotypic factors of β-thalassemia in Bangladeshi β-thalassemia/HbE patients.
The objective of this study was to investigate the polymorphism of heat shock protein 70 ( hsp 70) gene in the F 2 populations of KU–Phuphan black-bone chicken comparing with crossbreed native chicken and commercial layer chicken populations. Thirty female and twenty-five male KU–Phuphan black-bone chickens, and twenty-three for each crossbreed native chicken and commercial layer chicken were genotyped hsp 70 gene by PCR–RFLP technique. The hsp 70 gene showed two loci, locus C generated by Eae I and locus M created by Mme I. The locus C consisted of two alleles C 1 and C 2 providing three genotypes of C 1 C 1 , C 1 C 2 and C 2 C 2 . The allele and genotype frequencies of KU–Phuphan black-bone chicken populations were not different from other Thai indigenous breeds. The locus M consisted of two alleles M 1 and M 2 whereas the two genotypes of M 1 M 2 and M 2 M 2 were observed. The allele M 1 was low allele frequency and was only found in MB population whereas allele M 2 showed monomorphism for FB, BR and LA populations. The male KU–Phuphan black-bone chicken exhibited different M 1 M 2 genotype from other detected populations but the allele frequency of MB was less than other Thai indigenous chickens. The genotype frequency of candidate thermotolerance genotype C 1 C 2 / M 2 M 2 of both KU–Phuphan black-bone chicken populations was higher than other genotypes. According to the results, KU–Phuphan black-bone chickens showed variation genotypes especially for locus C and contained high genotype frequency of the candidate thermotolerance genotype. Therefore, the locus C and the candidate genotype of hsp 70 gene could be used as genetic marker related with heat tolerance for selection and breeding program of KU–Phuphan black-bone chicken to create heat tolerance chickens.
Most hepatitis C virus (HCV) infection becomes chronic diseases and predictably develops into liver cirrhosis or hepatocellular carcinoma. HCV can be classified into 7 genotypes (GT) and multiple subtypes (ST). HCV treatment guidelines focus on determining the accuracy of HCV genotyping and subtyping, before starting treatment in all HCV-infected individuals. Efficient HCV genotyping and subtyping have become essential tools for indicating the optimal treatment. Current routine assays for HCV genotyping are easy and quick turnaround, but apparently subtyping is limited to identification. This study evaluated the accuracy of HCV genotyping in a newly developed automated-massively parallel sequencing (MPS) technique of previously identified HCV-infected specimens. The results indicated that by using the reference method, MPS and a bioinformatic pipeline, the genotype and subtype had identical accuracy of 95.65%. Interestingly, MPS could detect the remaining 4.35% as mixed-HCV type infected specimens, while the reference method could not. This result indicated that the MPS technique was highly accurate in identifying the HCV genotype and mixed-type infection. In addition, MPS can detect resistance-associated variants (RAVs) simultaneously with the HCV genotyping, especially in HCV GT1-infected specimens. Since HCV GT1 has a highly frequent presence of pre-existing RAVs, with resistance associated to direct-acting antivirals (DAAs), this study found that up to 60% of HCV GT1 sample were RAVs. This discovery of important RAVs in the patient infected with HCV GT1 could be beneficial in guiding the therapy decisions while avoiding the use of some DAAs. Thereby, the opportunity for patients to access the best treatment is enhanced.
Cholangiocarcinoma (CCA)is one of the worst prognosis cancers. CCA usually presents in advanced stage according to its high metastatic property. Until present, the factors involving in CCA metastasis are still unclear. Estrogen has been revealed as a stimulator of the invasiveness of CCA. Cyclooxygenase-2 (COX-2), an estrogen targeted gene, associates with cancer invasion and metastasis. This study aims to investigate the role of COX-2 in estrogen-stimulated invasion of CCA cells. KKU-100 and KKU-M213 CCA cell lines were treated with 17β-estradiol (E2), as active form of estrogen, and NS398, a selective COX-2 inhibitor, and the invasive property of cancer cells was measured as well as the expression levels of some metastatic genes. The results indicated that E2 could stimulate CCA cell invasion and this process was inhibited by NS398. In addition, in E2-stimulated CCA cells, the levels of COX-2 and CD44 were increased. These results indicate the impact of COX-2-mediated CD44 expression in E2-driven CCA invasion. The application of using COX-2 inhibitor to attenuate E2-stimulated invasion of CCA is of great interest to propose in the effective management of the disease.
Familial adenomatous polyposis (FAP) is an autosomal dominant genetic disease that is characterized by multiple adenomatous polyps in the colon and rectum. Adenomatous polyposis coli gene ( APC ) mutations are the causative event for FAP. APC consists of 15 exons, which encode 2,843 amino acids. The APC protein acts as a tumor suppressor and plays a role in several cellular processes. Here we investigated APC mutations in 20 unrelated Thai patients with FAP using denaturing high-performance liquid chromatography followed by DNA sequencing. Twenty mutations were identified in this study. Three mutations were novel mutations, including one nonsense mutation (c.2560A>T), one deletion mutation (c.4634_4638delCAAAT) and one indel mutation (c.3746_3748delinsAA). Data from mutations in Thai patients with FAP will be useful for molecular genetics diagnosis and genetic counseling of patients with FAP.
Intra-articular glucocorticoid (GC) injection such as triamcinolone acetonide is an ultimate pharmaceutical treatment for knee osteoarthritis (OA) patients who do not respond well to other pharmaceutical treatments. Effects of GCs on homeostasis of cartilage’s extracellular matrix (ECM) are not conclusive. In this study, we investigated whether TA caused destruction to ECM by altering expression of genes involved in digestion of collagens and proteoglycans. Chondrocytes isolated from cartilages of 10 knee OA patients were treated with TA at 1 and 5 mg/ml. Reverse-transcription real-time polymerase chain reaction was used for evaluating expressions of genes involving ECM degradation ( matrix metalloproteinase ( MMP )- 1, MMP-3, MMP-13, ADAM with thrombospondin motives ( ADAMTS ) -5 and tissue inhibitor of matrix metalloproteinase ( TIMP ) -3 ). Statistical differences in relative-quantification expression levels between TA-treated chondrocytes and non-treated controls were found in MMP-3 (6.59 ± 5.2-fold for 1 mg/ml TA ( p = 0.022) and 5.43 ± 3.46-fold for 5 mg/ml TA ( p < 0.01)). Levels of MMP-1 , MMP-13 , ADAMTS-5 , and TIMP-3 were not significantly changed. TA-increased expressions of MMP-3 in chondrocytes might assist further degradation of cartilage and accelerate OA progression. Results from this study may assist physicians in TA treatment planning for OA patients.
Interspecific hybrids between Jatropha curcas × J. integerrima are potential genetic resources for jatropha improvement. However, their pollen is normally partially sterile. This study was conducted to determine pollen viability status and seed set of the hybrids. Pollen viability was estimated by staining with 1% acetocarmine. The results showed that all interspecific hybrids had pollen viability falling between their parents. The pollen viability of interspecific hybrid derived from a cross between J. curcas (high phorbol esters accession) × J. integerrima ranged from 47 % to 89 % with an average of 72 %. Similarly the viability of the hybrid J. curcas (low phorbol esters accession) × J. integerrima ranged from 44 % to 90 % with an average of 73 %. The hybrid with low pollen viability also set less seeds. This information is useful for plant breeders to select the pollen donor in interspecific breeding programs.
OSB1 gene encodes myc-type basic helix-loop-helix (bHLH) transcription factor which controls expression of several structural genes involving in anthocyanin biosynthesis in rice. In this study, the expression of OSB1 gene was investigated in young leaves and developing seeds of six rice varieties, including white (Taichung 65 and Sasanishiki), red (Sang Yod and Hom Mali Dang) and black (Khum and Lerm Poa), by RT-PCR. The OSB1 gene was expressed in both leaves and seeds of all six rice varieties. The full-length coding sequences of OSB1 genes were isolated from young leaves of white rice Sasanishiki and black rice Khum. Nucleotide sequence analysis revealed that Khum and Sasanishiki had open reading frames (ORF) of 1,767 and 1,725 bp, respectively which were 99–100% and 100% identical with OSB1 genes in black and white rice reported in GenBank database, respectively. It was found that OSB1 gene from white rice showed a 2-bp insertion in the 7 th exon that caused frameshift mutation and premature termination, leading to the truncation of 14 amino acids at C-terminus of the regulatory protein. Moreover, the amino acid substitution of T64M was found in white rice OSB1 protein, affecting the conserved N-terminal interacting domain, probably causing the non-function of the OSB1 gene in white rice. The OSB1 gene from black rice was active in anthocyanin biosynthesis, suggesting this gene might play an important role in anthocyanin pigmentation, especially in seeds. The cloned OSB1 genes will be further analyzed for gene functions to understand the regulation of anthocyanin biosynthesis in colored rice.
Piper betle Linn. (familyPiperaceae) producesvarieties of sesquiterpenevolatilecompoundsthat have valuable pharmacological activities. Unfortunately, the active compounds are in low amount in plants and their chemically production is still difficult. These make them insufficientusage. Biotechnology is the better choice for promotion of the compounds production. Hence, in this report, the inducers of sesquiterpenesynthase ( STS ) , the key enzyme of the sesquiterpene biosynthesis, were evaluated.Methyljasmonateatvarious concentration and time including 100, 200 and 300 µM was sprayed for 6 12 and 24 hr. and mechanical wounding was appliedon ten-week-old P.betle tissue culture. Afterwards, the STS transcript was investigated by Realtime-qPCR technique. The results showed that the highest upregulation of STS was detected after 100 µMMeJAfor 12 hr. application. Furthermore, wounding alsoinduced STS expression. In conclusion, there was probability that P.betle responded to environmental stress through methyljasmonate and sesquiterpeneproduction via sesquiterpenesynthase activation. บทคดยอ สารเซสควเทอรปนเปนสารอนทรยระเหยงายทพบไดในพล ถงแมวาสารในกลมนมฤทธทางชวภาพหลากหลายแตการใชประโยชนจากสารยงคงมอยจำกด เนองจากโครงสรางโมเลกลของสารซบซอน ทำใหการสงเคราะหโดยขบวนการทางเคมทำไดยาก นอกเหนอจากนนพชเองกสงเคราะหไดไมเพยงพอ การใชเทคโนโลยทางชวภาพเขามาชวย จงเปนทางเลอกสำหรบการเพมการผลตสารกลมนได งานวจยนจงไดทำการศกษาหาปจจยกระตนการแสดงออกของยน sesquiterpene synthase (STS) ซงเปนเอนไซมหลกในวถสงเคราะหสารเซสควเทอรปนในเนอเยอเพาะเลยงพล โดยฉดพนเนอเยอเพาะเลยงพลอาย 10 สปดาห ดวยสารเมทลจสโมเนท ซาลไซเลท ไคโตซาน เซลลเลส และการทำใหเกดบาดแผลบนใบ หลงจากนนทำการวดระดบทรานสครปของ STS ดวยวธ reverse transcription PCR (RT-PCR) และ real-time quantitative PCR (RT-qPCR) พบวาระดบทรานสครปของ STS สงทสด เมอพลไดรบ MeJA 100 µM เปนเวลา 12 ชวโมง นอกเหนอจากนนการทำใหเกดบาดแผลมผลกระตนการแสดงออกของยน STS ดวย การคนพบนแสดงใหเหนถงกลไกทพลใชตอบสนองตอสภาวะเครยดจากสงแวดลอม โดยการสงสญญาณผานเมทลจสโมเนทและการเพมการสรางสารเซสควเทอรปนดวยการกระตนเอนไซม sesquiterpene synthase ความรทไดจะเปนประโยชนตอการพฒนาวธการเพอเพมการผลตสารเซสควเทอรปนเพอนำไปประยกตใชตอไป
The objective of this study was to determine human bloodstains by duplex PCR analysis of cytochrome b gene ( Cyt b ). Two regions of Cyt b consisting of conserved and human specific regions were the targets of interest. The primers specific to these regions were designed and tested with DNA extracted from human and animal (dog, cat, pig, chicken and cow) blood samples. A PCR product of Cyt b conserved region (~309 bp) was found in both human and animal blood samples while a DNA fragment of human specific region (~412 bp) was seen only in human blood sample. Duplex PCR analysis was then carried out. Human blood could be discriminated from animal blood by the number of amplified fragment, two for human and one for animal blood samples. The duplex PCR analyses of mixed human and animal bloodstains were also investigated. Duplex PCR could determine human DNA even when the quantity of human blood was 100 fold less than that of animal blood. The effects of temperature and age of bloodstains on human blood determination were then examined. Human bloodstain samples were kept at different temperatures (25, 35, 45 and 55°C) for different periods of time (1, 7, 14 and 30 days). The results demonstrated that duplex PCR analysis is able to determine human bloodstains in all experimented conditions.
Autosomal dominant polycystic kidney disease (ADPKD) is the most common dominantly inherited kidney disease, being caused by mutations in PKD1 (85%) and PKD2 (15%). Approximately 25% of all PKD1 mutations are pathogenic missense, but additional 12.8% of those are reported as indeterminate or missense variants of uncertain significance (VUS), of which the deleterious nature are unclear in clinical practice. Functional studies to assess the impact of missense variants, particularly for PKD1 , are difficult due to the large size and ambiguous functions of the proteins, polycystins. A variety of in silico tools were developed to evaluate interspecies variations and biochemical impacts of amino acid substitutions of missense VUS. To evaluate the tool suitability for PKD1 and PKD2 , we applied additional 12 state-of-the-art web-based tools with their claimed superior performances based on different algorithms for the prediction in parallel with the three benchmark programs (PolyPhen2, SIFT and MutationTaster). A total of 15 tools were assessed in a gene-specific manner with PKD1 and PKD2 variants of known pathogenicity from the PKDB mutation database. We found that each of the genes had suitable predictions from different sets of tools. Combined uses of 3, 5, 8 and 12 tools with high performance in descending order (in which the benchmarks were excluded) gave consensus predictions for the selected nine VUS in PKD1 , except one VUS which might have a mild pathogenicity that only the use of 12 tools predicted differently. In this situation, using more numbers of tools might prevent the misinterpretation of milder VUS. Classification of the pathogenicity of the VUS in PKD1 and PKD2 became an essential part of molecular diagnosis of ADPKD and is useful for a clinical decision and would expand knowledge of mutation spectrum in ADPKD.
Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder, characterized by the degeneration of motor neurons of the spinal cord, thus leading to the deaths of newborns. More than 95% of SMA patients are caused by the absence of survival motor neuron 1 ( SMN1 ) gene exon 7, located on chromosome 5q13. The disease prevalence and carrier frequency of SMA in Thailand have not ever been reported. This research aims to explore the frequency of SMA carriers in whom, SMN1- exon 7 is heterozygously deleted and simultaneously determine the SMN2- exon 7 copy number in Thai population. This may also help to assess the prevalence of SMA in Thailand. We used a denaturing high-performance liquid chromatography (DHPLC)-based semi-quantitative multiplex-PCR technique to detect the copy number of SMN genes by comparing with two reference genes. The analysis was carried out using 1,000 DNA samples extracted from the dried blood spot (DBS) specimens leftover from the Thai National Neonatal Screening Program. Results indicated that the SMA carrier frequency in the Thai newborns is approximately 2.8% (1/36 cases). It can be extrapolated that the prevalence of SMA in Thai newborns caused by homozygous deletion of SMN1 exon 7 is 1 in 5,102 cases. Our findings indicated that SMA can be considered as a national health problem as the incidence found is close to what has been reported elsewhere. The results can also be utilized to establish a national program for clinical, prenatal diagnosis and genetic counseling to effectively prevent SMA in Thailand.
Three groups of Hansenula polymorpha fatty acid auxotrophic mutants were screened by their fatty acid requirement for growth. The mutants are saturated fatty acid auxotrophic mutants (Sfa - ), monounsaturated fatty acid auxotrophic mutants (Mfa - ) and polyunsaturated fatty acid auxotrophic mutants (Pfa - ) which required saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids, respectively. All Sfa - mutants could grow on the media supplemented with C14:0 but not grow on the media with neither C16:0 nor C18:0. By fatty acid analysis, two of the Sfa - mutants, S7 and S16, showed a significant difference in the fatty acid composition. S7 clearly defected in the production of C18:2Δ9,12 and C18:3Δ9,12,15, while S16 significantly accumulated medium-chain saturated fatty acids, C12:0 and C14:0. Mfa - and Pfa - mutants could grow on the media supplemented with monounsaturated fatty acids better than supplemented with polyunsaturated fatty acids. The mutants obtained in this study could be used as tools for studying in genes involved in the fatty acid synthesis and biological effect of fatty acids.
ABSTRACT Long interspersed element-1 (LINE-1) are retrotransposible element. Recent studies showed that gene containing LINE-1s are regulated by epigenetic regulation of intragenic LINE-1s in cancer and in embryonic development. Moreover, intragenic LINE-1 may possess a regulatory function under physiologic condition. We hypothesized that female sex steroids could altered expressions of genes containing LINE-1. Here we analyzed the influence of intragenic LINE-1on gene expression during menstrual phases by using expression array data from public database (Gene Expression Omnibus (GEO), (http://www.ncbi.nlm.nih.gov/geo). Gene expression levels in early-, mid- and late- secretory phase were compared with those in proliferative phase. Genes containing intragenic LINE-1were highly and significantly up-regulated in early-secretory phase as compared to those in proliferative phase in both GSEs tested (GSE6364 with p< 1.05E-10; OR = 1.62; 95% CI = 1.88–1.40 and GSE4888 with p<2.22E-10; OR=1.61; 95% CI=1.87-1.39). While genes containing intragenic LINE-1 were significantly up-regulated in both mid and late-secretory phase as compared to those in proliferative phase (GSE4888 with p< 5.66E-03; OR=1.24; 95% CI = 1.44–1.06 and p<1.14E-04; OR=1.42; 95% CI=1.69-1.19. Whereas genes containing intragenic LINE-1 were found to be significantly down-regulated only in mid-secretory phase as compared to those in proliferative phase. (GSE6364 p<2.98E-02; OR=1.15; 95% CI=1.31-1.01 and GSE4888 p<6.22E-04; OR=1.25; 95% CI=1.41-1.10). Together, these data suggested that intragenic LINE-1 could play a role in regulation and expression of sex steroid regulated genes during menstrual cycle.
S. curtisii Hook.f. roots are used as herbal insecticide due to its production of alkaloids, specifically stemocurtisine. Industrial production of insecticide from S. curtisii Hook.f. is problematic because of difficulties in distinguishing S. curtisii Hook.f. roots from those of other species. DNA analysis of Stemona spp. roots using Dellaporta et al .’s (1983) extraction method and the HAT-RAPD technique with 20 primers indicated that 10 primers, including B-01, B-05, B-07, N-13, N-18, O-10, OPAG 07, OPAG 14, OPAI 08 and OPAJ 09, produced DNA bands capable of distinguishing S. curtisii Hook.f. from other species. รากของตนหนอนตายหยากชนด Stemona curtisii สามารถนำมาผลตสารกำจดศตรพชไดเนองจากมสารแอลคาลอยดชนด stemocurtisine แตการผลตสารกำจดศตรพชในระดบอตสาหกรรมพบวามปญหาในการจดซอวตถดบเพราะไมสามารถแยกความแตกตางของรากชนด S. curtisii กบรากหนอนตายหยากชนดอนไดดวยตาเปลา ในการทดลองนจงใชเครองหมายโมเลกลจากเทคนค high annealing temperature random amplified polymorphic DNA (HAT-RAPD) ในการจำแนกชนดของตนหนอนตายหยากจากสวนราก โดยวธทเหมาะสมสำหรบสกดดเอนเอจากสวนรากคอ วธของ Dellaporta et al. (1983) เมอนำดเอนเอมาตรวจสอบดวยเทคนค HAT-RAPD โดยใชไพรเมอรจำนวน 20 ไพรเมอร พบวาม 10 ไพรเมอร ไดแก B-01, B-05, B-07, N-13, N-18, O-10, OPAG 07, OPAG 14, OPAI 08 และ OPAJ 09 ทแสดงลายพมพดเอนเอของ S. curtisii แตกตางจากหนอนตายหยากชนดอน
Phytophthora capsici Leonian is one of the most important pathogens, which destruct the pepper crop ( Capcicum sp .) in worldwide. The inheritance of Phytophthora resistant gene is complex inheritance, known as quantitative trait loci (QTL). The major QTL is located on chromosome 5. In this study, bulked segregant analysis (BSA) approach was used for discovery of polymorphic SNP markers in double haploid population derived from highly resistant Capsicum annuum accession Criollo de Morelos – 334 and susceptible variety Jindu. The linkage maps were constructed and QTL analysis was performed. The QTL with the highest LOD score, detected on chromosome 5, explained 31% of the phenotypic variance with a LOD score of 11. The Second QTL, designated as minor QTL, which was found on chromosome 8, explained 11.9% of the phenotypic with LOD score of 3.36. The nearest marker, which was located on major QTL and minor QTL were SNP_421 and SNP_694, respectively. Therefore, two SNP markers were the best candidate markers that linked to QTL of Phytophthora resistant gene.
Induced pluripotent stem cells (iPSCs) have a great potential for disease modeling and therapeutic applications. One of the most common genetic diseases found in South East Asia is thalassemia. β-Thalassemia is an inherited single gene disorder caused by reduction or absence of β-globin chain production. The purpose of this study is to generate mouse iPSCs with β-thalassemia background. Mouse embryonic fibroblast (MEF) cells were obtained from transgenic β-thalassemic mouse, transduced with lentiviral vector carrying 4 transcription factors, Oct4, Klf4, Sox2 and c-Myc (OKSM), and cultured on a feeder layer. The embryonic stem (ES)-like colonies were manually picked up and expanded. The generated mouse iPSCs were similar to mouse embryonic stem cells (mESCs) in morphology, alkaline phosphatase activity and pattern of pluripotent gene expression and able to form teratoma containing tissues derived from all three germ layers. Therefore, we reported here an establishment of β-thalassemic mouse iPSCs which might be used for further disease modeling and therapeutic applications.
Prenatal diagnosis (PND) is a technique to detect chromosome abnormalities in fetus before birth. The techniques currently used for PND are conventional karyotyping and fluorescent in situ hybridization (FISH). Both techniques still have limitations, e.g., a high error rate of detection. The purpose of this study was to evaluate the efficiency of KaryoLite BACs-on-Beads (KL-BoBs™) as a routine diagnostic assay in a clinical application for PND by comparison with conventional karyotyping. A total of 37 amniotic fluid cell samples from 18 patients were used to validate KL-BoBs™ technique. The results of the KL-BoBs™ were 100% concordant with karyotyping for trisomy 18, trisomy 21, triple X, normal XX and normal XY. In conclusion, our results indicated that KL-BoBs™ had high concordance with conventional karyotying for prenatal diagnosis to detect chromosome status in amniotic fluid cells. Moreover, using an appropriate reference could reduce the coefficient of variation (CV) and increase accuracy of interpretation.
