Observational experiments on Bartlett's microsomes in human cells

Summary

M.L. Barr et al. were the first to find a darkly stained vesicle in the interphase nucleus of nerve cells in female cats, but not in male cats. Since this vesicle was related to sex and the number of X chromosomes, it was called X chromatin, or Barrbody. Later, the same microsomes were found in other female mammalian cells and in many cells of human females. It is now generally accepted that X chromatin is formed when one of the two X chromosomes undergoes heteroconcentration during interphase and is usually inactivated. The inactivated chromosomes are randomized, and the inactivated state makes the genetic effect of the two copies of genes carried by the two X chromosomes in females almost equal to that of the one copy of genes carried by one X chromosome in males, resulting in a kind of dosage compensation effect, which is unique to the maintenance of the uniformity of gene expression in male and female organisms. Source: Laboratory Course in Genetics

Operation method

basic program

Principle

In the case of sex chromosome aberrations, two Bachmann's vesicles can be observed in female somatic cells with XXX karyotype (Figure 12-1), i.e., except for one X chromosome, the rest of the X chromosome is inactivated, and Bachmann's vesicles can also be observed in male somatic cells with 47,XXY karyotype. The study of Barthelia can help to reveal the regulation mechanism of X-linked genes, the evolution of sex chromosomes, and explain the symptoms of patients with sex chromosome aberrations.

Materials and Instruments

Human oral mucosa cells, hair root sheath cells, etc.
Methanol Glacial acetic acid 50%, 70%, 75%, 95% ethanol and anhydrous ethanol Ethyl ether 50% acetic acid solution 5 mol L hydrochloric acid Phosphoric acid buffer 1% aqueous thiokurin solution
Microscopes (including fluorescence microscopes) Constant temperature water baths Tongue depressor plates Tweezers Slides and coverslips

Move

1. Sampling and preparation. Oral mucosal cells: first let the subject rinse his mouth with water for several times, in order to remove the debris in the mouth as far as possible, then scrape the buccal mucosa of the subject's mouth with a medical tongue depressor, smear the scrapings on a slide, and then dry it.

Hair root sheath cells: One hair with hair root sheath was taken from the subject and placed on a clean slide. Add a drop of 50% acetic acid solution to the root and leave it for 5 min. After the hair is softened, scrape off the root tissue with a clean needle, remove the hairs and dry them, separate the scraped tissues evenly with a needle and dry them.

2. Place the preparation in methanol-glacial acetic acid (3:1) fixative for 20 min.

3. Put the slide into 95%, 70%, 50% ethanol and distilled water for 2~3min each time.

4. Hydrolyze the slides in 5 mol/L hydrochloric acid for about 5 s. During this process, RNA was broken down, and RNA-rich vesicles were stained by Corydalis sulfate. This step is very critical, acid digestion is not enough to remove RNA, and excessive acid digestion will damage DNA.

5. Rinse with distilled water 2~3 times, 10~15s each time.

6. Corydaline staining for 10～20min, rinse with distilled water, drying can be examined by microscope. If the staining is too deep, it can be differentiated in 95% ethanol for about 30s, if it is too light, it can be re-stained. Human hair root sheath cells can be directly stained with aqueous solution of Corydalis sulfate, transferred to 75% ethanol for about 30s, gently swung, and then taken out to dry.

100 cells with large nuclei, clear staining and clear outlines were selected and the frequency of X chromatin was counted. X chromatin was located on the inner side of the nuclear membrane of the cells, with clear outlines of plano-convex, triangular or ovoid shape, dark blue in color and 1～1.5 μm in diameter. at least 15% of the cells in the positive smears contained X chromatin, and generally not more than 30%～35%. X chromatin may occasionally be observed in smears from males, but the frequency is less than 2%.

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