阅读说明：本技术 一种优化的适用于绵羊染色体二元流式分选的染色体悬液制备方法 (Optimized preparation method of chromosome suspension suitable for binary flow sorting of sheep chromosomes ) 是由 邓学梅 姚延珠 于 2020-07-22 设计创作，主要内容包括：本发明公开了一种优化的适用于绵羊染色体二元流式分选的染色体悬液制备方法。本发明提供了一种绵羊染色体悬液的制备方法,包括如下步骤：将同步化至M期的绵羊成纤维细胞依次进行过滤、低渗处理和裂解,得到绵羊染色体悬液。本发明的试验证明了经细胞筛过滤后收集的M期绵羊成纤维细胞所制备的绵羊染色体悬液经流式细胞仪分析后,所得绵羊二元流式核型图比未经过过滤的M期绵羊成纤维细胞制备的染色体悬液所得绵羊二元流式核型图具有更高的分辨率,更清晰。为一种优化的适用于绵羊二元流式分选的染色体悬液制备的方法。(The invention discloses an optimized preparation method of chromosome suspension suitable for binary flow sorting of sheep chromosomes. The invention provides a preparation method of a sheep chromosome suspension, which comprises the following steps: and sequentially filtering, hypotonic treating and cracking the sheep fibroblasts synchronized to the M stage to obtain the sheep chromosome suspension. The experiment proves that after the sheep chromosome suspension prepared from the M-phase sheep fibroblasts collected after filtration by the cell sieve is analyzed by the flow cytometer, the obtained sheep binary flow karyotype chart has higher resolution and is clearer than the sheep binary flow karyotype chart prepared from the chromosome suspension prepared from the M-phase sheep fibroblasts which are not filtered. Is an optimized method for preparing chromosome suspension suitable for sheep binary flow sorting.)

1. A method for preparing a sheep chromosome suspension comprises the following steps:

1) filtering the sheep fibroblast synchronized to the M stage to obtain filtered cells;

2) preparing a sheep chromosome suspension by using the filtered cells.

2. The method of claim 1, wherein: the pore size of the filtration was 40 μm.

3. The method according to claim 1 or 2, characterized in that: the filtration was performed using a 40 μm cell sieve.

4. A method according to any one of claims 1-3, characterized in that:

the preparation of the sheep chromosome suspension by using the filtered cells comprises the following steps:

2) -A, hypotonic treatment of said filtered cells to obtain hypotonic treated cells;

2) and B, cracking the hypotonic treated cells to obtain a sheep chromosome suspension.

5. The method according to any one of claims 1-4, wherein:

the sheep fibroblasts synchronized to the M phase are prepared as follows: synchronizing the in vitro sheep fibroblasts to the S phase through TDR repression, and synchronizing the cells synchronized to the S phase to the M phase through Nocodazole treatment to obtain the sheep fibroblasts synchronized to the M phase.

6. A suspension of sheep chromosomes produced by the method of any one of claims 1 to 5.

7. Use of a method according to any one of claims 1 to 5 or a suspension of sheep chromosomes according to claim 6 in the preparation of a binary flow karyotype chart for sheep.

8. A method for improving the definition of a sheep binary flow karyotype chart comprises the following steps:

1) prepared by the method of any one of claims 1 to 5 to obtain a suspension of sheep chromosomes;

2) and detecting the sheep chromosome suspension by using a double-laser flow cytometer to improve the definition of the sheep binary flow karyotype chart.

9. Filtering the sheep fibroblast by a 40-micron cell sieve, and then synchronizing the sheep fibroblast to the M stage to prepare the sheep chromosome suspension;

or, the application of the 40 μm cell sieve in the preparation of sheep chromosome suspension;

or the application of a 40-micron cell sieve, a hypotonic solution and a cell lysate in preparing the sheep chromosome suspension.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an optimized preparation method of chromosome suspension suitable for binary flow sorting of sheep chromosomes.

Background

The subunits formed by the nuclear genome of humans, animals and plants are called chromosomes. When metaphase chromosomes are dispersed in suspension, they can be classified using flow cytometry based on light scattering and fluorescence parameters. Karyotypes obtained by analyzing chromosomes by flow cytometry are called flow karyotypes. The flow karyotype chart of animal chromosomes is a suspension of chromosomes of a certain species, and after staining with two fluorescent dyes (usually Hoechst 33258-AT specific binding and chromomycin A3-GC specific binding) which are specifically bound to AT and GC respectively, chromosomes with different sizes and AT/GC contents can be separated by flow cytometry analysis, and the obtained flow karyotype chart is called a binary flow karyotype chart. However, chromosomes with small differences in size and GC content are not easily separated. The so-called binary flow karyotype is generated according to the fluorescence intensity of the two dyes, and the purity of the flow sorting chromosome can reach more than 95% under high-quality samples and good conditions.

Chromosome flow sorting plays an important role in nuclear genome structure analysis and special chromosome and abnormal chromosome analysis. Chromosomes sorted by flow cytometry have been used in genomics research such as DNA hybridization, DNA libraries, physical mapping, and chromosome sequencing. Flow sorting of chromosomes has become an attractive and powerful tool for genomic studies of chromosomes, due to their ability to isolate individual chromosomes in high purity and in large quantities.

Flow-based classification of sheep chromosomes was reported as early as 1992, and only the first three large chromosomes and the other five chromosome clusters could be resolved. Sheep were acquired in 1997 with high resolution streaming karyotypes in which almost all chromosomes were isolated and identified. However, the application of sheep chromosome flow sorting is rarely reported in the future. This is probably because the preparation conditions of the chromosome suspension are unstable, and it is difficult to obtain a stable and clear sheep binary flow karyotype chart.

The resolution of the binary flow karyotype chart is related to a number of factors, such as the hardware facilities and detailed equipment usage parameters of the flow cytometer, and the sample quality of the chromosome suspension.

The quality of chromosome suspension preparation is critical to chromosome flow cytometry sorting. High quality chromosome samples contain a large amount of single chromosomes in a suitable chromosome separation buffer, but contain fewer nuclear fragments and clumps of aggregated chromosomes. Mammalian chromosome suspensions are typically prepared from cells in the M phase of the cell cycle. Therefore, the growth state of the cells and the treatment mode of the M-phase cells can influence the quality of the preparation of the chromosome suspension, thereby influencing the definition of the final binary flow karyotype chart.

Disclosure of Invention

In the process of binary flow sorting of sheep, the invention finds that the preparation quality of the sheep chromosome suspension has sensitive influence on the definition of the finally generated sheep binary flow karyotype chart, thereby optimizing a certain factor of the preparation of the chromosome suspension.

The invention aims to provide an optimized method for preparing chromosome suspension suitable for sheep binary flow sorting.

The preparation method of the sheep chromosome suspension provided by the invention comprises the following steps:

1) filtering the sheep fibroblast synchronized to the M stage to obtain filtered cells;

2) preparing a sheep chromosome suspension by using the filtered cells.

In the above method, the pore size of the filtration is 40 μm.

In the above method, the filtration is performed using a 40 μm cell sieve.

The preparation of the sheep chromosome suspension by using the filtered cells comprises the following steps:

2) -A, hypotonic treatment of said filtered cells to obtain hypotonic treated cells;

2) and B, cracking the hypotonic treated cells to obtain a sheep chromosome suspension.

The above lysis was performed using PAB solution (15mM tris, 2mM EDTA, 0.5mM EGTA, 80mM KCl, 3mM dithiothreitol, 0.25% (volume fraction) triton X-100, 0.2mM spermidine, 0.5mM spermidine, balance water, pH 7.5);

the hypotonic treatment is to collect cells which can pass through a filter screen, centrifuge the cells for 5 minutes at 289 Xg, and add 15ml of hypotonic solution for hypotonic treatment for 30 minutes at room temperature (25 ℃); the hypotonic solution is as follows: 75mM KCl, 0.2mM spermidine, 0.5mM spermidine, 10mM MgSO4 & 7H2O, balance water, pH 8.0.

In the method, the sheep fibroblast synchronized to the M phase is prepared according to the following method: synchronizing the in vitro sheep fibroblasts to the S phase through double TDR repression, and synchronizing the cells synchronized to the S phase to the M phase through Nocodazole treatment to obtain the sheep fibroblasts synchronized to the M phase.

The sheep chromosome suspension prepared by the method is also the protection scope of the invention.

The application of the method or the sheep chromosome suspension in the preparation of the sheep binary flow karyotype chart is also within the protection scope of the invention.

The invention also aims to provide a method for improving the definition of the sheep binary flow karyotype chart.

The method provided by the invention comprises the following steps:

1) the sheep chromosome suspension is obtained by adopting the preparation method;

2) and detecting the sheep chromosome suspension by using a double-laser flow cytometer to improve the definition of the sheep binary flow karyotype chart.

The improvement of the definition of the sheep binary flow karyotype chart is that the definition of the sheep binary flow karyotype chart of the sheep chromosome suspension prepared by the method (including filtering) is higher than that of the sheep binary flow karyotype chart of the sheep chromosome suspension prepared without the filtering step in the method.

The application of the sheep fibroblast synchronized to the M phase after being filtered by a 40-micron cell sieve in the preparation of the sheep chromosome suspension is also within the protection scope of the invention;

or, the application of the 40 μm cell sieve in the preparation of the sheep chromosome suspension is also the protection scope of the invention;

alternatively, the application of 40 μm cell sieve, hypotonic solution and cell lysate in the preparation of sheep chromosome suspension is also within the scope of the present invention.

The method provided by the invention comprises the following steps: and collecting the sheep fibroblasts synchronized to the M phase, filtering by using a cell sieve, and cracking the filtered M phase sheep fibroblasts by using a PAB solution, wherein the obtained sheep chromosome suspension is suitable for flow sorting, and a sheep binary flow karyotype chart which is clearer than a sheep binary flow karyotype chart prepared from unfiltered M phase cells can be obtained.

In the above method, the M-stage sheep fibroblasts are suppressed to S-stage by bisthymidine (TDR,2mM) and then suppressed to M-stage by nocodazole (final concentration: 100 ng/ml).

In the method, the chromosome suspension prepared from the M-phase sheep fibroblasts filtered by the 40-micron cell sieve is clearer than the chromosome suspension prepared from the M-phase cells which are not excessive, and the sheep binary flow karyotype is obtained.

Experiments prove that after the sheep chromosome suspension prepared from the M-phase sheep fibroblasts collected after filtration by the cell sieve is analyzed by a flow cytometer, the obtained sheep binary flow karyotype chart has higher resolution and is clearer than the sheep binary flow karyotype chart prepared from the chromosome suspension prepared from the M-phase sheep fibroblasts which are not filtered. Is an optimized method for preparing chromosome suspension suitable for sheep binary flow sorting.

Drawings

FIG. 1 shows isolated and purified sheep primary fibroblasts (200X).

FIG. 2 shows the synchronization effect of the S phase of the sheep skin fibroblasts; (A) cell cycle profiles following dual TDR repression; (B) cell cycle profiles of normal control cells without any treatment.

FIG. 3 shows the effect of M-phase synchronization of sheep skin fibroblasts; (A) is a cell cycle distribution map after M-phase synchronization; (B) cell cycle profiles of normal control cells without any treatment.

FIG. 4 is a sheep binary flow karyotype; (A) and (B) a sheep binary flow karyotype chart generated after chromosome suspension prepared from M-phase cells filtered by a 40-micron cell sieve is sorted by a flow cytometer; (C) and (D) a sheep binary flow karyotype chart generated by sorting chromosome suspensions prepared from M-phase cells without filtration treatment by a flow cytometer.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The complete medium composition in all the following examples was: DMEM medium (Gibico, cat. No. 11995065) containing 10% (volume fraction) FBS and 1% (volume fraction) Penicilin-Streptomycin-Glutamine (100X) (Gibco, cat. No. 10378016).