5.Rapid detection of SOX2 gene by primed in situ labeling.
Xi LUO ; Xianping DING ; Lin CHEN ; Qiang QUAN
Chinese Journal of Medical Genetics 2012;29(3):289-292
OBJECTIVETo rapidly detect SOX2 gene using primed in situ labeling (PRINS).
METHODSHuman peripheral blood samples were cultured using an optimized method. Sequence of the SOX2 gene was amplified in situ with biotin-labeled specific primers and processed with a tyramide signal amplification (TSA) biotin system. Subsequently, fluorescence-stained signal was detected by streptavidin-Texas red. For the control group, MCF-10F cells were transfected with Lentivirus hSox2.
RESULTSBy VideoTesT-FISH software analysis, the long arm of chromosome 3 in the experimental group showed a specific red fluorescence signal, whilst the control samples showed no specific signals for SOX2. Transfected MCF-10F cells showed various efficiency of SOX2 gene integration.
CONCLUSIONPRINS utilizes a highly sensitive in situ PCR technique combined with fluorescence labeled oligodeoxynucleotides can synthesize probes in situ, thus greatly reducing the cost of probe and time for detection. It can facilitate identification and classification of induced pluripotent stem cells, and has many potential applications in this prospect.
Humans ; In Situ Hybridization, Fluorescence ; methods ; Male ; Primed In Situ Labeling ; methods ; SOXB1 Transcription Factors ; chemistry
6.Detection of the SRY gene by primed in situ labeling.
Yong NIE ; Xianping DING ; Li DENG ; Ping WEI ; Huan WANG
Chinese Journal of Medical Genetics 2008;25(6):701-703
OBJECTIVETo establish a primed in situ labeling (PRINS) technique which can be more effective in detection of single copy gene.
METHODSOn the basis of traditional PRINS, new reagents and procedures, such as TaqStart antibody, four primers of the sex determining region Y (SRY) gene and TSA(TM) Biotin System were included in detection of the SRY gene. Meanwhile, fluorescence in situ hybridization(FISH) to detect the SRY gene was used as control.
RESULTSFifty metaphases were scored. PRINS labeling showed signals for the SRY on the Y chromosome at band Yp11.3 in all metaphases. These signals were as distinct as that from results of FISH.
CONCLUSIONThis improved method is ideal for rapidly localizing single copy genes and small DNA segments. And PRINS is a cost- and time-effective alternative to FISH.
Gene Dosage ; Genes, sry ; genetics ; Humans ; In Situ Hybridization, Fluorescence ; Male ; Metaphase ; genetics ; Primed In Situ Labeling ; methods
8.The application of an in situ karyotyping technique for mesenchymal stromal cells: a validation and comparison study with classical G-banding.
Sang Mee HWANG ; Cha Ja SEE ; Jungeun CHOI ; Seon Young KIM ; Qute CHOI ; Jung Ah KIM ; Jiseok KWON ; Si Nae PARK ; Kyongok IM ; Il Hoan OH ; Dong Soon LEE
Experimental & Molecular Medicine 2013;45(12):e68-
The cytogenetic analysis of mesenchymal stromal cells (MSCs) is essential for verifying the safety and stability of MSCs. An in situ technique, which uses cells grown on coverslips for karyotyping and minimizes cell manipulation, is the standard protocol for the chromosome analysis of amniotic fluids. Therefore, we applied the in situ karyotyping technique in MSCs and compared the quality of metaphases and karyotyping results with classical G-banding and chromosomal abnormalities with fluorescence in situ hybridization (FISH). Human adipose- and umbilical cord-derived MSC cell lines (American Type Culture Collection PCS-500-011, PCS-500-010) were used for evaluation. The quality of metaphases was assessed by analyzing the chromosome numbers in each metaphase, the overlaps of chromosomes and the mean length of chromosome 1. FISH was performed in the interphase nuclei of MSCs for 6q, 7q and 17q abnormalities and for the enumeration of chromosomes via oligo-FISH in adipose-derived MSCs. The number of chromosomes in each metaphase was more variable in classical G-banding. The overlap of chromosomes and the mean length of chromosome 1 as observed via in situ karyotyping were comparable to those of classical G-banding (P=0.218 and 0.674, respectively). Classical G-banding and in situ karyotyping by two personnel showed normal karyotypes for both cell lines in five passages. No numerical or structural chromosomal abnormalities were found by the interphase-FISH. In situ karyotyping showed equivalent karyotype results, and the quality of the metaphases was not inferior to classical G-banding. Thus, in situ karyotyping with minimized cell manipulation and the use of less cells would be useful for karyotyping MSCs.
Azure Stains
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Chromosome Banding/*methods
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Humans
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In Situ Hybridization, Fluorescence/*methods
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Karyotyping/*methods
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Mesenchymal Stromal Cells/*cytology
9.Advances in preimplantation genetic diagnosis for chromosomal translocation carrier.
Yueqiu TAN ; Guangxiu LU ; Luyun LI
Chinese Journal of Medical Genetics 2002;19(1):76-78
Chromosomal translocation is a kind of common chromosomal abnormality. The carriers with chromosomal translocation could have more chance of normal pregnancy with the help of fluorescence in situ hybridization(FISH). This is a review aimed at analyzing the meiosis types of the translocation chromosome. The strategy of preimplantation genetic diagnosis for the carriers with chromosomal translocation is also discussed.
Humans
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In Situ Hybridization, Fluorescence
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methods
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Meiosis
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genetics
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Preimplantation Diagnosis
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methods
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Translocation, Genetic
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genetics
Result Analysis
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