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Immortalized quality-control cell bank for chromosome karyotype analysis and construction method thereof

A technology of chromosome karyotype and construction method, which is applied in the field of chromosome karyotype analysis, immortalized quality control cell bank and its construction, and can solve the problems of no feasible quality control substances

Inactive Publication Date: 2014-09-24
翁炳焕
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Problems solved by technology

[0010] In order to solve the problem that there is no feasible quality control substance in prenatal cytogenetic diagnosis, the inventor proposes the present invention

Method used

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  • Immortalized quality-control cell bank for chromosome karyotype analysis and construction method thereof

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Embodiment Construction

[0016] 1. Protocells: The protocells used are derived from the remaining primary or subcultured living adherent growth after routine chromosomal disease laboratory diagnosis due to the needs of clinical diagnosis and treatment, which have been diagnosed as difficult, rare chromosomal abnormalities, and diagnostic reports are issued. Cells, the cell types are fetal amniotic fluid, villi tissue and other cells.

[0017] 2. Extraction of SV40 large T antigen DNA (SV40LTag DNA): ① SV40 DNA enzyme digestion: buy commercially available SV40 lyophilized powder or SV40 plasmid containing large T antigen gene, dissolve in appropriate amount of H 2 In O or TE buffer, add 2uL 10× digestion buffer and 18uL H 2O, add restriction endonuclease BamH I (1-5U / ugDNA), incubate at 37°C for 1h, heat at 75°C for 15min, inactivate the enzyme, add 5uL electrophoresis loading buffer (also by adding 0.5mol / LEDTA) Stop the reaction and prepare for electrophoresis. ②SV40 DNA electrophoresis: Take elect...

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Abstract

The invention relates to an immortalized quality-control cell bank for chromosome karyotype analysis in the medicine field and a construction method thereof. The invention is mainly characterized in that the construction method comprises: taking residual attached primary or sub-culture living cells which are used for chromosomal disease diagnosis as needed by clinical diagnosis and treatment and are definitely diagnosed to have difficult chromosome abnormalities, introducing, through transgenosis technology, SV40LTag-pcDNA3.1(-) recombinant plasmid constructed by pcDNA3.1(-) connected by T4DNA ligase and digested by BamHI, and SV40LTag DNA amplified by PCR and separated by agarose gel electrophoresis, allowing recon to integrate with the cell DNA, screening immortalized cells integrated with recons by G418, performing subculture amplification and living cell cryopreservation, then extracting several respectively-prepared cells with different diseases, mixing the cells according to a required ratio for quality control so as to convert each original cell with a single chromosome abnormality into a cell with a plurality of easy-to-misdiagnose chromosome abnormalities with different ratios. Therefore, the difficulty and complexity for differential diagnosis and chromosome chimera diagnosis are increased; samples are easily available; and waste residual cells are converted into effective quality-control materials.

Description

technical field [0001] The invention relates to an immortalized quality control cell bank for chromosome karyotype analysis and a construction method thereof, which is mainly used in cytogenetic diagnostic laboratories in the medical field for technical assessment of chromosome karyotype analysis, indoor quality control and external quality assessment. Background technique [0002] Chromosomes are the genetic material in the nucleus. Humans have 23 pairs of chromosomes, 22 of which are autosomes, and 1 pair is the sex chromosome that determines the sex of men and women. Genes carrying genetic information on chromosomes, of which DNA accounts for more than 90%, and RNA content varies with cell cycle and growth, generally accounting for 1% to 10%. [0003] Chromosomal structural abnormalities, abnormal numbers, and mosaicism are clinically manifested as chromosomal diseases, which are common birth defects, such as congenital stupidity, primary amenorrhea, and hermaphroditism. ...

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Application Information

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IPC IPC(8): C40B40/02C40B50/06C12N5/10C12N15/85C12Q1/68C12Q1/02
Inventor 翁炳焕
Owner 翁炳焕
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