KR100631338B1 - Relative Amount Analysis of Telomeres on Chromosomes - Google Patents

Abstract

The present invention comprises the steps of culturing the fibroblasts of domestic animals to obtain a sample of the cell division medium phase; Treating the sample with a telomeric DNA probe to conjugate the chromosome of the sample with the telomeric DNA probe; And it relates to a method for measuring the telomeres content of the chromosome characterized in that the chromosome and the telomeric DNA probe is observed by a microscope and the relative content of telomeres from the observed image.

Chromosomes, telomeres, splicing

Description

Relative Amount Analysis of Telomeres on Chromosomes

Figure 1 is a photograph showing the mid-phase distribution of cell division of the bovine chromosome conjugated with the telomeric DNA probe.

Figure 2 is a photograph showing the karyotype of the small chromosome conjugated with the telomeric DNA probe.

Figure 3 is a photograph showing the mid-phase distribution of cell division of the porcine chromosome conjugated with the telomeric DNA probe.

Figure 4 is a photograph showing the karyotype of the pig chromosome conjugated with the telomeric DNA probe.

The present invention relates to a method for measuring the telomere content of an animal chromosome, the method comprising the steps of: ⒜ culturing fibroblasts of an animal to obtain a sample of a cell division medium phase; (V) subjecting the sample of step iii to a telomeric DNA probe to conjugate the chromosome and the telomeric DNA probe of the sample; And (iii) a sample of step iii under a microscope and measuring the relative content of telomeres from the observed images.

Telomere is a complex of DNA-proteins present in eukaryotic cells. It consists of a tandem repeated DNA sequence (TTAGGG) and a specific protein at the end of a chromosome. The length of telomeres increases as cell division progresses. When the telomeres are shorter than a certain length, cell death is aborted. Telomerase, on the other hand, is a ribonucleoprotein that is involved in the synthesis of telomeric DNA and plays a role in supplementing the loss of telomeric DNA at the ends of chromosomes.

Many recent studies have reported that the role of telomeres is intrinsically responsible for the stability of chromosomes and is associated with the aging of cells or the development of cancer (Anmed et al. J. Am. Geriatr. Soc., 2001, Vol. 49 ( 8), p. 1105; Cottliar et al. Medicina, 2001, Vol. 61, p. 335; Multani et al. Neoplasia, 2000, Vol. 2 (4), p. 339; Pathak et al. Int. J. Oncology, 2002, Vol. 20 (3), p.637; Robinson, MO Genet. Eng., 2000, Vol. 22, p.209; Sohn et al. Exptl. Cell Res., 2002, Vol. 279 (2 ), p.271). In normal cells, it is known that there is a loss of about 50 to 200 bp of telomeric DNA per cell cycle, and below a certain length, spontaneous death of spontaneous cell death (apoptosis) due to disruption is induced (Greider et al. Cell, 1985, Vol. 43, p. 405).

Therefore, studies on the relationship between the length of telomeres, the activity of telomerase, and cellular aging have been actively conducted. In particular, many studies have been reported in humans and rodents.

In humans, the expression of telomerase appears to be vigorous in fetal tissue but little in somatic cells after neonatal life (Meeker et al. A review Biochemistry, 1997, Vol. 62 (11), p. 1323). However, their expression continues in newly dividing cells, such as germ cells and stem cells, and cancer cells that continue to divide indefinitely (Ulaner et al. Mol. Hum. Repord., 1997, Vol. 3). (9), p. 769). Although most organs in the human body show a significant decrease in telomerase with age, the short-lived animals such as rodents (mouse, rats), unlike humans, do not develop telomerase after birth. Expression is reported to be persistent (Yamaguchi et al. Exptl. Cell Res., 1998, Vol. 242, p. 120). This means that as age increases, telomeres become shorter without the influence of telomerase and act as a different mechanism from humans.

In addition, a number of studies on the production of somatic clones using nuclear transfer have recently been conducted. As is well known, sheep (Wilmut et al. Nature, 1997, Vol. 385, p.810) and cattle (Cibelli et al. Sci) , 1998, Vol. 280, p. 1256) and successive successes in livestock, including pigs (Onishi et al. Sci., 2000, Vol. 289, p. 1188). However, Dolly, the first cloned sheep, was euthanized because of complications due to severe premature ejaculation. This unexpected aging of somatic clones has attracted interest in intracellular telomeres, especially in the case of cloned sheep Dolly, the controversy and controversy over these theories suggesting that short telomeres of donor cells are directly responsible for individual aging. Controversial views are actively discussed (Betts et al. Proc. Natl. Sci. USA, 2001, Vol. 98, p. 1077; Miyashita et al. Biol. Reprod., 2002, Vol. 66, p. 1649; Shiels et al. Nature, 1999, Vol. 399, p. 316; Tian et al. Nat. Genet., 2000, Vol. 26, p.272).

On the other hand, as described above, various studies on telomeres and telomerases related to aging are being progressed, and their functions and roles are being revealed. However, telomeres on livestock are relatively inadequate. In particular, little analysis of the distribution of chromosomal telomeres has been reported, since the length of telomeres can vary between species, ages, tissues within the same individual, and even between the same intracellular chromosomes. This is because distribution patterns are difficult to suggest (Hemann et al. Cell, 2001, Vol. 107, p.67). However, the presentation of telomere karyotypes and chromosomal distribution patterns for normal sexual axis are the basic markers that are the basis for telomere or telomerase studies.

Since a method of measuring and analyzing the length of a telomeric restriction fragment (TRF) using a Southern blot as a conventional telomere quantitative analysis method (Gan et al. Pharma Res., 2001, Vol. 18) (12), p. 1655), the telomere content of most animal tissues and cells, including cloned individuals, is presented by TRF analysis (Fradiani et al. Biochimie., 2004, Vol. 86, p.7; Miyashita et al. Biol.Reprod., 2002, Vol. 66, p. 1649).

However, in the TRF assay, DNA loss can occur during blotting, and in the case of extremely long telomeres such as mice, measurement errors can occur, and above all, the analysis is complex and time consuming. do.

In addition, the TRF assay can only measure the total telomere content in the cell, so it is impossible to analyze the distribution of telomeres in each chromosome.

Therefore, the present inventors have experimentally completed the analysis of the content of telomeres in livestock chromosomes using quantitative fluorescence in situ hybridization (Q-FISH) to more easily measure the relative content of telomeres. An attempt was made to develop a method.

Therefore, the present invention comprises the steps of culturing fibroblasts of domestic animals to obtain a sample of the cell division medium phase;

(V) subjecting the sample of step iii to a telomeric DNA probe to conjugate the chromosome and the telomeric DNA probe of the sample; And

Step VII provides a method for determining the telomeres content of a chromosome, characterized by observing the specimen of step VII under a microscope and measuring the relative content of telomeres from the observed images.

The present invention relates to a method for measuring the content of telomeres present in the livestock chromosome, ⒜ culturing the fibroblasts of the livestock to prepare a sample in the mid-cell division; (V) subjecting the sample of step iii to a telomeric DNA probe to conjugate the chromosome and the telomeric DNA probe of the sample; And a method of measuring the telomeres content of the chromosome, characterized by observing the specimen of step VII under a microscope and measuring the relative content of telomeres from the observed images.

The livestock used in the present invention is a mammal or a poultry, the mammal may be cattle, pigs, horses, sheep, dogs, cats, rabbits, poultry may use chickens, ducks, turkeys, quails, preferably cattle or It is good to use pigs.

In addition, the method for obtaining and culturing fibroblasts from the livestock used in the present invention is to chop the tissue of the livestock, and after treatment with a chelating agent that can separate the fibroblasts from the tissue, the tissue treated with the chelating agent Can be cultured in a culture medium consisting of several nutrients. Trypsin-EDTA solution may be used as the chelating agent, and fetal bovine serum, penicillin-streptomycin, triiodothyronine, and triiodothyronine may be used as a culture medium. , T3), insulin (Insulin), hydrocortisone (Hydrocortisone), glutamine (Glutamine), adenine (Adenine) or transferrin (Transferrin) and the like can be used.

On the other hand, in order to obtain the cultured fibroblasts as a cell division medium-phase specimen, it can be obtained by treating with a mitosis inhibitor before the end of culture, and separating only the cultured fibroblasts, followed by centrifugation.

The present invention is characterized by subjecting the obtained cellular medium-phase sample to a telomeric DNA probe to hybridize the chromosome and the telomeric DNA probe of the sample.

The telomeric DNA probe used in the present invention is characterized in that the fluorescent labeling material is bound. The fluorescent labeling material is intended to be displayed when the telomeres of the chromosomes to be measured of the specimen are combined with the telomeric DNA probe, Cy-3 (Cyanine-3); Rhodamine; Amino-methylcoumarin acetic acid (AMCA); Fluorescein; And Texas Red, but any one of the groups may be used. Preferably, a telomeric DNA probe labeled Cy-3 is used.

In addition, the telomeric DNA probe used in the present invention may use a general DNA probe consisting of DNA only, preferably PNA (peptide nucleic acid). Since the PNA is a base of the nucleic acid is bound to the peptide backbone (peptide backbone) and the base of the peptide is arranged at intervals similar to the nucleic acid skeleton, the PNA can also selectively bind to recognize the complementary base sequence of the nucleic acid, chromosome When combined with telomeres of ions, the ion repulsion is less than that of general DNA-DNA binding.

 In addition, it will be readily appreciated by those skilled in the art that the preparation of the telomeric DNA probe used in the present invention may be prepared by using a sequence complementary to the telomer of the chromosome to be measured.

Method for measuring the telomeres of chromosomes according to the present invention by observing the combination of the telomeres and telomeres DNA probe of the chromosome to be measured in the sample under a microscope, by measuring the relative length of the telomeres from the observed image chromosome The telomere content of can be measured.

As the microscope used in the present invention, it is preferable to use a fluorescent microscope capable of observing a fluorescent labeling material and obtaining a observed image as a digital image.

In addition, the method for measuring the telomere content in the present invention can obtain the image shown through the microscope as a digital image, it is possible to measure the telomere content for each chromosome using an image analysis program. That is, after obtaining a digital image of the cell division mid-phase from the microscope, the relative content of telomeres is measured by analyzing the ratio of the color luminosity between the sites where the telomeres and the telomeric DNA probes are bound to each chromosome and the sites where they are not. can do.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the present invention is not limited by the following examples.

<Example>

Example 1

Test Axis and Test Materials

In the present invention, 10 males of 32-36-month-old Holstein and 18-month-old Lendace sow were used in the experimental farm of Texas A & M University. Ear tissue was biopsyd from the subjects and fibroblast cultures were performed from these tissues.

Example 2

Chromosome Isolation and Sample Preparation Using Fibroblast Culture

Fibroblast culture was performed by removing several tissues from the ears of the Holstein species male or Renrace species sows of Example 1, washing several times with PBS solution, finely slicing in sterile conditions, and 0.25% trypsin in the fine tissue. After treatment with trypsin) -EDTA (Gibco., Invitrogen Corp., USA), 15% fetal bovine serum was prepared using medium-199 (Medium-199, Gibco., Invitrogen Corp., USA) as a base medium. , Gibco., Invitrogen Corp., USA), 17.5 penicillin-streptomycin (Gibco., Invitrogen Corp., USA) in the mixed culture solution added trypsin-EDTA treated 37.5 ℃, Incubated under 5% CO ₂ conditions.

When the cells in culture form a monolayer on the entire bottom of the culture flask, 0.25 cells were treated with trypsin-EDTA to separate the cells, and a certain amount of the separated cells were divided into culture flasks and passaged three times. .

In addition, in order to obtain a cell specimen of the cell division medium phase, the culture specimen was treated with 0.2 µg / ml of colcemid (Gibco., Invitrogen Corp., USA) 50 minutes before the end of the culture, incubated for 50 minutes, and then scraped. Cells were recovered by scraper and centrifuged at 200 μg.

Storage treatment was carried out for 6 minutes at 37 ℃ to 0.4% potassium chloride solution (Sigma Chem., USA) and 0.4% sodium citrate solution (Sigma Chem., USA) 1: 1 in the cell suspension subjected to the centrifugation. , The fixation treatment was carried out by repeating the Carnoy's solution (methanol: acetic acid = 3: 1) three times in the stored cell suspension.

Sampling was prepared by dropping 3-4 drops of the fixed cell suspension on a slide stored in the refrigerator for one day.

Example 3

Determination of Telomere Content in Chromosomes

Cy-conjugated peptide nucleic acid human telomere probe (DA-3, DAKO Corp., USA), a fluorescently labeled telomeric DNA probe, was prepared on the sample prepared in Example 3. Quantitative fluorescence junction verification was performed. That is, after the slide sample was pretreated with RNase A, the conjugated solution containing the fluorescently labeled telomeric DNA probe was dropped on the slide, covered with a cover slip, and sealed with rubber cement. The sealed slides were treated for 10 minutes at 72 ° C. to denature the probes and samples, and bonded for 1 hour at 38.5 ° C., and then washed with 2 × SSC solution and PN (sodium phosphate and nonidet P-40) buffer. For background staining, 2,4-diamidine-2-phenylindole (DAPI) solution was used, and the expression of fluorescence conjugate was triple-band pass filter. Observed with a fluorescence microscope (Microphot-FAX, Nikon, Japan) attached to, at least 50 chromosome chromosomes of cell division was obtained by digital image. Each stored chromosome image was analyzed for telomeric DNA content for each chromosome using an image analysis program (MetaMorph Image System, Universal Imaging Co., U.S.A.).

Content analysis was analyzed by measuring the red / blue intensity ratio (Red / Bule intensity ratio). That is, the background staining of the whole chromosome was treated with DAPI solution (blue), and Cy-3 (red) was attached to the telomer conjugate probe to analyze the ratio of its color luminosity to calculate the relative content of chromosome telomeres.

Cell division of the small chromosome and the karyotype of the experimental results in the experimental results are shown in Figures 1 and 2, the telomeres content of the small chromosome is shown in Table 1.

In addition, the cell division mid-phase distribution and karyotype of the porcine chromosome is shown in Figures 3 and 4, the telomeres content of the porcine chromosome is shown in Table 2.

The value is the mean value ± standard deviation.

^{The a} p value represents the mean value of the short arm and long arm by the t-test.

*, ** Mean values of short arm and long arm have significant differences at p <0.05 * and p <0.01 **.

The value is the mean value ± standard deviation.

^{The a} p value represents the mean value of the short arm and long arm by the t-test.

^b Chromosome 6 has a relative content of 10.72 ± 2.12 with telomeres inserted at the bottom of the centromere.

*, ** Mean values of short arm and long arm have significant differences at p <0.05 * and p <0.01 **.

As shown in Figs. 1 and 2, the conjugation pattern of the distinct telomeric DNA probe was found at the distal ends of all bovine chromosomes, and the conjugation in the form of two distinct dots at each distal end appeared. The presence or absence of (p-arm) was clearly confirmed.

In addition, as shown in Table 1, the overall chromosome content of bovine chromosome was analyzed to be 33.8%, and the telomere content of each bovine chromosome was significantly different among chromosomes (p <0.01). The distribution of telomeres between q-arms showed little difference except for chromosome 9, but the overall content of telomeres at the distal end of the arm was higher than that of the distal end.

On the other hand, in the case of chromosome 9, the long arm terminal showed a higher content of telomeres than the short arm terminal, which showed a three-fold difference in the telomer content of the short arm. The chromosome with the highest relative content of telomeres was the Y chromosome.

As shown in Fig. 3 and 4, the porcine chromosome also showed the conjugation pattern of the telomeric DNA probe at the end of the chromosome in the same manner as the bovine chromosome. However, chromosome 6 of the porcine chromosome showed interstitial telomere at the lower end of the chromosome, and the distribution of telomeres was the same on the homologous chromosome 6 of all the cell divisions.

In addition, as shown in Table 2, in the case of porcine chromosomes, there was a difference in the telomer content of each chromosome, and unlike the small chromosome, there was also a difference in the telomer content between short and long arm in the same chromosome. The total telomeres on pigs were 12.3%, which was lower than that on bovine chromosomes.

The present invention is a method for measuring the telomeres content of the chromosome can more easily and efficiently determine the telomeres karyotype of the livestock and the distribution of telomeres on the chromosomes.

Claims (4)

(A) separating and culturing fibroblasts from livestock and then treating and culturing the mitosis inhibitor before terminating the culture, and isolating the cultivated fibroblasts to obtain a sample of mid-cell division; (Iii) conjugating the chromosome and telomeric DNA probe of the sample by treating the sample of step iii with a telomeric DNA probe labeled with a fluorescent labeling substance; And 표본 A method of measuring telomeres for each chromosome, characterized by observing the sample of step 관찰 under a fluorescence microscope and measuring the relative content of telomeres for each chromosome from the observed images. The method according to claim 1, wherein the livestock is mammalian or poultry. The method according to claim 1, wherein the telomeric DNA probe is PNA. The method of claim 1, wherein the telomer content of the chromosome is measured by quantitative fluorescence conjugation method.

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