• Journal of Animal Reproduction and Biotechnology
• /
• v.35 no.4
• /
• pp.323-328
• /
• 2020

Direct injection of genome editing tools such as CRISPR/Cas9 system into developing embryos has been widely used to generate genetically engineered pigs. The approach allows us to produce pigs carrying targeted modifications at high efficiency without having to apply somatic cell nuclear transfer. However, the targeted modifications during embryogenesis often result in mosaicism, which causes issues in phenotyping founder animals and establishing a group of pigs carrying intended modifications. This study was aimed to establish a genomic PCR and sequencing system of a single blastomere in the four-cell embryos to detect potential mosaicism. We performed genomic PCR in four individual blastomeres from four-cell embryos. We successfully amplified target genomic region from single blastomeres of 4-cell stage embryo by PCR. Sanger sequencing of the PCR amplicons obtained from the blastomeres suggested that PCR-based genotyping of single blastomere was a feasible method to determine mutation type generated by genome editing technology such as CRISPR/Cas9 in early stage embryos. In conclusion, we successfully genotyped single blastomeres in a single 4-cell stage embryo to detect potential mosaicism in porcine embryos. Our approach offers a simple platform that can be used to screen the prevalence of mosaicism from designed CRISPR/Cas9 systems.

• KSBB Journal
• /
• v.20 no.4
• /
• pp.323-327
• /
• 2005

RT-PCR is an useful method to investigate the expression of target gene as detection tools. Although RT-PCR is the powerful detection method for tissues, it was difficult to amplify the target gene product using the single cell. To clarify the expression level of the genes related to Parkinson's disease (PD), I performed the laser dissection of single cell from Substantia nigra. I examined the mRNA expression level in the dopaminergic neuron isolated from the PD patients by the single cell RT-PCR method. It is known that tyrosine hydroxylase (TH), DOPA decarboxylase (DDC) are involved in biosynthesis of the catecholamine such as dopamine. Little has been known about the gene expression features of these enzymes in single dopaminergic neuron. I could detect the specific gene products in single cell level. The different expression was observed in PD-related gene products from the single neuron of PD patients. Interestingly, TH gene expression was significantly decreased with comparing the ratio of decrease in other PD-related genes. Hence, I represented data that indicate the RT-PCR method described in this report is an effective method in detecting a specific single-cell mRNA level related with diseases.

• Animal cells and systems
• /
• v.13 no.3
• /
• pp.351-356
• /
• 2009

Ciliates are undoubtedly one of the most diverse protozoans that play a significant role in ecology. However, molecular examination, based on comparing the DNA sequences, has been done on a limited number of the species. Because most ciliates are uncultivable and their population sizes are often too small, it is usually difficult to obtain sufficient genomic DNA required for PCR based experiments. In the present study, we evaluated the effectiveness of four commercial DNA extraction procedures that extract high quality genomic DNA from a single ciliate cell. It was discovered that RED Extract-N-$Amp^{TM}$ PCR kit is the best method for removing PCR-inhibiting substances and minimizing DNA loss during purification. This method can also amplify more than 25 reactions of PCR. In addition, this technique was applied to single cells of 19 species belonged to 7 orders under 5 classes that isolated from mixed natural populations. Their small subunit ribosomal DNA (SSU rDNA) was successfully amplified. In summary, we developed a simple technique for the high-yield extraction of purified DNA from a single ciliate cell that may be more useful for rare ciliates, such as tiny and uncultivable marine microbes.

• Proceedings of the KSAR Conference
• /
• 2005.06a
• /
• pp.18-18
• /
• 2005

• Asian Pacific Journal of Cancer Prevention
• /
• v.17 no.7
• /
• pp.3065-3069
• /
• 2016

Single nucleotide polymorphism (SNP) detection has been used extensively for genetic association studies of diseases including cancer. For mass, yet accurate and more economic SNP detection we have optimized tetra primer amplification refractory mutation system polymerase chain reaction (ARMS PCR) to detect three SNPs in the cytochrome P450 2E1 (CYP2E1) gene locus; i.e. rs3813865, rs2070672 and rs3813867. The optimization system strategies used were (1) designing inner and outer primers; (2) determining of their optimum primer concentration ratios; and (3) determining of the optimum PCR annealing temperature. The tetra primer ARMS PCR result could be directly observed using agarose gel electrophoresis. The method succesfully determined three SNPs in CYP2E1 locus, the results being consistent with validation using DNA sequencing and restriction fragment length polymorphisms (RFLP).

• Korean Journal of Agricultural Science
• /
• v.49 no.4
• /
• pp.795-807
• /
• 2022

The development and commercialization of industrial genetically modified (GM) organisms is actively progressing worldwide, highlighting an increased need for improved safety management protocols. We sought to establish an environmental monitoring method, using real-time polymerase chain reaction (PCR) and propidium monoazide (PMA) treatment to develop a quantitative detection protocol for living GM microorganisms. We developed a duplex TaqMan quantitative PCR (qPCR) assay to simultaneously detect the selectable antibiotic gene, ampicillin (AmpR), and the single-copy Escherichia coli taxon-specific gene, D-1-deoxyxylulose 5-phosphate synthase (dxs), using a direct cell suspension culture. We identified viable engineered E. coli cells by performing qPCR on PMA-treated cells. The theoretical cell density (true copy numbers) calculated from mean quantification cycle (Cq) values of PMA-qPCR showed a bias of 7.71% from the colony-forming unit (CFU), which was within ±25% of the acceptance criteria of the European Network of GMO Laboratories (ENGL). PMA-qPCR to detect AmpR and dxs was highly sensitive and was able to detect target genes from a 10,000-fold (10^-4) diluted cell suspension, with a limit of detection at 95% confidence (LOD_95%) of 134 viable E. coli cells. Compared to DNA-based qPCR methods, the cell suspension direct PMA-qPCR analysis provides reliable results and is a quick and accurate method to monitor living GM E. coli cells that can potentially be released into the environment.

• Journal of the Korean Society of Marine Environment & Safety
• /
• v.24 no.7
• /
• pp.967-972
• /
• 2018

The genus Chattonella belonging to the class raphidophyceae, is a harmful algal bloom species. Recently, its occurrence has been increasing and expanding along the Korean coast. Species identification of the genus Chattonella only by morphological observation is difficult due to the lack of rigid cell walls. In this study, the morphological characteristics and genetic affinity of Chattonella sp. isolated from Deungnyang Bay in 2017 were examined. We carried out single-cell isolation from field samples then sequenced three different areas using the single-cell PCR method: 1) parts of ribosomal operon, the large subunit (LSU) of the rDNA, 2) the chloroplast-encoded subunit psaA of Photosystem I, and 3) rbcL encoding the large subunit of the Rubisco gene. The cells were morphologically very similar to the general genus Chattonella ($74.0{\pm}10.1{\mu}m$ in length, $33.1{\pm}3.6{\mu}m$ in width). The three partial gene sequences were insufficient to justify distinction at the species rank. However, they clustered at 99-100 % sequence similarity with C. marina, C. marina var. antiqua and C. marina var. ovata.

• Animal cells and systems
• /
• v.15 no.3
• /
• pp.251-258
• /
• 2011

Ciliates are considered one of the most diverse protozoa and play significant roles in ecology. For successful taxonomic study of these microscopic eukaryotes, a staining procedure is necessary, due mainly to intrinsic difficulties in recognizing characteristics from living cells. Although molecular taxonomy has been used to resolve the ambiguities associated with traditional morphology-based taxonomy, extraction of genomic DNA from stained ciliate cells is not available yet. In the present study, we describe a method to extract genomic DNA from a single protargol-impregnated euplotid cell. By using $HgCl_2$ as a fixative and modulating the exposure time of bleach solution in the protargol impregnation, high-quality genomic DNA can successfully be extracted from a stained single cell with minimal loss of morphological integrity. This technique will contribute to the effectiveness of combined approaches of molecular and morphological taxonomy from single ciliate cells.

• Ocean and Polar Research
• /
• v.32 no.3
• /
• pp.247-254
• /
• 2010

Single cell PCR analysis and light and scanning electron microscopic techniques were utilized to identify free living bivalve larvae in the coastal waters of Tae-an, on the west coast of Korea. Through DNA sequencing, venerid clam larvae were isolated and identified as Ruditapes philippinarum (99% similarity) and Meretrix lusoria (99%). Under microscopic observation, the D-veliger stage of R. philippinarum exhibited symmetrical shoulder angles and an elliptical ventral form. In contrast, M. lusoria displayed asymmetrical shoulder angles and a round ventral form in the umbonal stage. Size of the R. philippinarum larvae was $156{\pm}22{\mu}m$ in length, $126{\pm}12{\mu}m$ in height, $92{\pm}14{\mu}m$ in width with a length: height ratio of 1.23. Meretrix lusoria was $202{\pm}44{\mu}m$ in length, $161{\pm}35{\mu}m$ in height, $96{\pm}38{\mu}m$ in width with a length: height ratio of 1.25. Experimental results indicate that morphological and molecular characteristics provide evidence for the larval identification of these two venerid clam larvae species in nature.

• BMB Reports
• /
• v.32 no.6
• /
• pp.529-534
• /
• 1999

Asymmetric PCR and single-strand conformation polymorphism (SSCP) methods were combined to analyze human leukocyte antigen (HLA)-DRB allele polymorphism. Asymmetric PCR amplification was applied to generate single-stranded DNA (ssDNA) using the nonradioactive oligonucleotide primers desinged for the polymorphic exon 2 region. The conformational differences of ssDNAs, depending on the allele type, were analyzed by nondenaturing polyacrylamide gel electrophoresis and visualized by ethidium bromide staining. The ssDNAs were clearly separated from double-stranded DNA without interference and obviously migrated depending on their allele type. This method was applied to the genomic DNA either from homozygous or from heterozygous cell lines containing the DR4 allele as template DNA using DR4-specific primers, and satisfying results were obtained. Compared to the standard PCR-SSCP method, this asymmetric PCR-SSCP method has advantages of increased speed, reproducibility, and convenience. Along with PCR-SSP or sequence-based typing, this method will be useful in routine typing of HLA-DRB allele.