CN106979938A - It is a kind of to carry out the method that destination gene expression is quantitatively detected using flow cytometer - Google Patents

Abstract

The invention provides a method for quantitative detection of target gene expression by using a flow cytometer. In cells expressing the target gene to be studied, the red fluorescent protein gene is knocked in; in the reading frame of the target gene, the green fluorescent protein is knocked in. Protein gene; the red and green fluorescent protein genes and the target gene are expressed simultaneously, but they form their own proteins independently of each other; the formed cells that can express the red and green fluorescent proteins at the same time are used to monitor the external factors that affect the expression of the target gene Screening; use flow cytometry to quantitatively detect the strength of the green fluorescent signal of the cells, and then use the red fluorescent signal value for homogenization, so as to quantitatively compare the influence of external factors on the expression of the target gene, and quantitatively detect the impact on the expression of the target gene external factors. The method can simultaneously compare the difference in the expression level of a specific gene in cells under various treatment conditions, and perform high-sensitivity screening of factors affecting gene expression in a relatively high throughput and within a relatively short period of time.

Description

A method for quantitative detection of target gene expression using flow cytometry

technical field

The invention relates to a method for quantitative detection of target gene expression by using a flow cytometer, and belongs to the technical field of molecular biology.

Background technique

Flow cytometry is a technique for one-by-one, multi-parameter, rapid qualitative and quantitative analysis or sorting of single cells or biological particles in a fast linear flow state. It has the advantages of fast detection speed, many measurement parameters, and a large amount of collected data , comprehensive analysis, high sorting purity, and flexible methods. Flow cytometry (FCM) is based on flow cytometry as the core technology, integrating physics, chemistry, biology, and multidisciplinary theories such as lasers and computers. The technical equipment integrated with technology is widely used in medical testing, cell biology, drug screening and other multidisciplinary fields. With the advancement of technology and the popularization of instruments, its application fields are constantly being expanded.

Based on the detection principle of flow cytometry, the cells to be detected are made into a single cell suspension after being fluorescently labeled, arranged in a single row under the coating of the sheath fluid, and pass through the area to be detected in turn, and the fluorescently stained cells are irradiated by the laser , produce scattered light and excited fluorescence, which can respectively reflect the size of the cell and the complexity of intracellular particles after being collected by the detector, as well as the information of the labeled particles inside the tested cell, these optical signals are converted into electrical signals, and are detected Transfer to computer to convert into data files to be saved and processed. The data is processed to derive information such as cell size, viability, DNA, RNA content, and cell cycle.

In immunological research, flow cytometry can analyze the proportion of lymphocytes, the proportion of T, B and NK cells in lymphocytes, T cell subsets and cell phenotypes; using DNA fluorescent dye propidium iodide ( Propidium Iodide (PI), combined with the DNA in the cell, can reflect the DNA content in the cell, thereby determining the mitotic cycle of the cell; by detecting whether the DNA content is abnormal or not, it can also distinguish between normal cells and tumor cells; Cytometers can also be used for drug screening to help screen out anti-tumor drugs by detecting the effects of drugs on cell apoptosis and other phenotypes. The above screening principle is based on the qualitative detection function of flow cytometry, which distinguishes different cell types according to size, shape, DNA content and antibody type. On the other hand, flow cytometry can also perform quantification according to the intensity of fluorescent signals, such as Chen Yang et al. (Chen Yang, Xu Rong, Si Shuyi, He Qiyang (2011), Flow Cytometry High-throughput Drug Screening The establishment and application of technology, Qilu Pharmaceutical Affairs, 30(1):1-2) The red fluorescent dye rhodamine was used as the substrate of tumor cell multi-drug resistance protein P-glycoprotein, and the cells after drug action were detected by flow cytometry. The accumulation of rhodamine in the high-throughput screening of drugs that can reverse the multidrug resistance of tumor cells, 8 positive compounds were screened from 8000 compounds. In this method, in addition to being a quantified marker, the fluorescent dye also has the function of a substrate, which is directly related to the function being studied, so it does not have general applicability to other phenotype studies.

The detection of gene expression is of great significance to the analysis of gene function and the search for genes related to specific phenotypes and diseases. Finding the factors that affect the expression of functional genes has an important guiding role in analyzing the causes of related diseases and finding suitable treatment methods. Widely used gene expression detection methods include Northern blot, RT-PCR, expression profile chip, etc. based on the mRNA level, and Western blot, ELISA, mass spectrometry, etc. based on the protein level. There are advantages and disadvantages in each aspect.

Contents of the invention

The technical problem to be solved in the present invention is to provide a method based on quantitative detection of flow cytometry for gene expression research in cells, which can simultaneously compare cells under various treatment conditions (such as multiple compounds, etc.), The difference in the expression level of a specific gene can be used for high-sensitivity screening of factors affecting gene expression in a relatively high throughput and a short period of time.

In order to solve the above technical problems, the technical solution of the present invention is to provide a method for quantitative detection of target gene expression using flow cytometry, characterized in that the steps are:

Step 1: In the cells expressing the target gene to be studied, knock in the red fluorescent protein gene; in the reading frame of the target gene, knock in the green fluorescent protein gene; make the red and green fluorescent protein genes and the target gene Simultaneously expressed, but independently form their own proteins; the formed cells that can express both red and green fluorescent proteins are used to screen external factors that affect the expression of the target gene;

Step 2: After external factors enter the cells formed in step 1, the expression of the target gene will be affected by up-regulation or down-regulation, and the impact will be reflected in the expression of green fluorescent protein; the quantitative detection of the cell by flow cytometry The strength of the green fluorescent signal is then normalized by the value of the red fluorescent signal, so as to quantitatively compare the influence of the external factors on the expression of the target gene, and quantitatively detect the external factors that affect the expression of the target gene.

Preferably, in step 1, external factors include small molecular compounds, microRNA, and protein expression vectors.

Preferably, in the step 1, the coding sequence of EGFP is inserted into the downstream of the coding sequence of the target gene in frame, and the coding sequence of the polypeptide P2A capable of self-cleavage during translation is introduced between the two, then the red and green two The two fluorescent proteins can be translated simultaneously but independently of each other, and the upstream and downstream transcription and translation regulatory regions of the target gene are completely preserved at the gene level, and the expression level of EGFP can faithfully reflect the impact of external factors on the expression of the target gene.

According to the method provided by the present invention, in selected mammalian cells (mainly mouse cells) that can normally express the target gene, a two-color fluorescent reporter system is constructed, with red fluorescence as the background color, and the tdtomato expression reading frame is passed through the homologous Recombination site-specific insertion into the mouse genome at the Rosa26 site; the green fluorescent protein gene indicating the expression level of the target gene is inserted in the same frame downstream of the last exon of the target gene, and a self-translation process is introduced between the two. The coding sequence of polypeptide P2A was cleaved, so that the target gene and EGFP were translated simultaneously but two independent proteins were obtained. After the cells with the dual fluorescent reporter system are constructed, they can be used to determine the factors that affect the expression of the target gene. The up-regulation and down-regulation of the target protein expression by these additional factors can be carried out by detecting the intensity of green fluorescent protein with flow cytometry. Quantitative evaluation, since the evaluation of each compound is based on the average value of a large number of cells of the same type (usually 1.0*10 ⁴ ), it can accurately detect small differences between samples; while detecting the green fluorescent signal, each The red fluorescence signal in each cell can also be detected at the same time, and the red fluorescence intensity can be used as an internal reference to normalize the detection error between each sample and improve the reliability of the detection result.

In the field of cell model construction for gene expression regulation, the present invention has two originalities:

First, build a fluorescent reporter system that directly reflects the expression of the target gene, and add fluorescent protein as an internal reference to homogenize the results; red fluorescence is used as the background and internal reference to normalize the cell state of cells that have been treated differently, and green fluorescence The strength of the gene can directly indicate the level of expression of the target gene;

Second, use flow cytometry to quantitatively detect the signal of the fluorescent protein, compare the green fluorescent signal value after the homogenization of the red fluorescent signal between samples, and obtain the screening result; the fluorescent signal of the cell is quantitatively detected by the flow cytometer, The expression level of the target gene can be compared between cell samples treated with different conditions, so as to screen out the factors that affect the expression of the target gene, including small molecular compounds, microRNA, protein, etc.

The method provided by the present invention constructs a two-color fluorescence reporting system, uses flow cytometry to detect the fluorescence signal of cells, and quantifies the fluorescence intensity of the same batch of cells, and compares the difference in fluorescence values in control cell samples under different treatment conditions, thereby Determine the effect of different treatment conditions on the expression of specific genes. This method can simultaneously compare the difference in the expression level of specific genes in cells under various treatment conditions, and perform high-sensitivity screening of factors affecting gene expression in a relatively high throughput and in a short period of time. It can be used in applied and basic research fields such as screening of compounds targeting drug targets, gene function research, and signaling pathway research.

Description of drawings

Fig. 1 is a schematic diagram of the expression method of red fluorescent protein in cells;

Figure 2 is a schematic diagram of the method for inserting the green fluorescent protein gene;

Fig. 3 is the flowchart of constructing the two-color reporter system for Kiss1 gene;

Figure 4 is a Western-blot verification of the expression of the green fluorescent protein gene;

Fig. 5 is the impact of flow cytometry detection of estradiol on Kiss1 gene expression; (A) is the flow histogram of negative control group and estrogen treatment group; (B) is the EGFP mean of negative control group and estrogen treatment group Histogram of fluorescence intensity; (C) is the histogram of average fluorescence intensity of tdtomato in negative control group and estrogen treatment group; (D) is the histogram of relative fluorescence intensity of EGFP/tdtomato in negative control group and estrogen treatment group;

Figure 6 is the reverse transcription fluorescent quantitative PCR verification of the influence of estradiol on the expression of Kiss1 gene;

Figure 7 is a Western-blot verification of the impact of estradiol on the expression of kisspeptin and green fluorescent protein;

Figure 8 is the flow cytometry detection of the impact of rapamycin on Kiss1 gene expression; (A) is the negative control group and rapamycin treatment group flow histogram; (B) is the negative control group and rapamycin The histogram of EGFP average fluorescence intensity in the treatment group; (C) is the histogram of the average fluorescence intensity of tdtomato in the negative control group and rapamycin treatment group; (D) is the EGFP/tdtomato in the negative control group and rapamycin treatment group Relative fluorescence intensity histogram;

Fig. 9 is a comparison of the effect of compounds on the expression of Kiss1 gene by flow cytometry.

detailed description

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

A method for quantitative detection of target gene expression using flow cytometry, specifically as follows:

In cells expressing the target gene to be studied, use Madisen et al. (Madisen L, Zwingman TA, Sunkin SM, et al (2010). A robust and high-throughput Cre reporting and characterization system for the whole mouse brain, 13(1 ): 133-40.) The constructed Ai9-dtomato plasmid knocks in the red fluorescent reporter gene, knocks in the green fluorescent protein gene in the reading frame of the target gene, so that it can be expressed simultaneously with the target gene but independently form their own proteins, The cells that can express both red and green fluorescent proteins can be used to screen for external factors that affect the expression of target genes, including small molecular compounds, microRNA, protein expression vectors, etc. After entering the cells through incubation, transfection, etc., the expression of the target gene may be up-regulated or down-regulated, and these effects will be reflected in the expression of green fluorescent protein. Quantitatively detect the strength of the green fluorescent signal of these cells by flow cytometry, and then use their red fluorescent signal value to normalize, so that the influence of these factors on the expression of the target gene can be quantitatively compared at a high throughput. Screen out compounds that affect the expression of the target gene, microRNA and protein, etc.

The coding sequence of EGFP is inserted into the downstream of the coding sequence of the target gene in frame, and the coding sequence of the polypeptide P2A capable of self-cleavage during translation is introduced between the two. These two proteins can be translated simultaneously but independently of each other, and in At the gene level, the upstream and downstream transcription and translation regulatory regions of the target gene are completely preserved, and the expression level of EGFP can faithfully reflect the impact of external factors on the expression of the target gene. The application of red and green double fluorescence improves the accuracy of the comparison between the two factors.

Use the flow cytometer to directly detect the signal of the fluorescent protein. The detection amount of each type of cell is 10,000, and 10,000 detection values can be obtained. The average value is used for comparison between samples, and effective statistical analysis and allows for reliable comparisons of small differences between samples. The detection by the traditional microplate reader is to quantify the overall fluorescence value of the cells in a cell culture well. If there are 10,000 cells in each well, only one value can be obtained, so more Only biological replicates can be used for statistical analysis, and the sensitivity is also low. The superiority of the method of the present invention can be seen.

The monoclonal cells expressing the dual fluorescent reporter system obtained through G418 and flow cytometry screening can be used for subsequent studies on the regulation of target gene expression. First, the cells are treated with known compounds that can up-regulate or down-regulate the expression of the target gene, microRNA or hormones, etc., as positive and negative controls. After 24 hours of treatment, the cells were digested into single cells with trypsin, fixed with 4% paraformaldehyde, and detected by flow cytometry, and the cells with similar morphology (cell size, granularity) after treatment were selected, and 10,000 of them were analyzed. The average green fluorescence value Ag and the average red fluorescence value Ar of each cell were compared with the negative and positive treatment groups by the ratio Ag/Ar of the two signals of the treatment group to preliminarily confirm the regulatory effect of the test factor on the target gene.

The following will be described with specific embodiments.

1. Construction of two-color fluorescent reporter system

Using the above method to construct a reporter system for Kiss1 gene in GT1-7 cells, the flow chart is shown in Figure 3.

2. Identification of two-color fluorescent reporter system

After screening by drugs and flow cytometry, monoclonal cells that can express both green and red fluorescent proteins were obtained, and a successful reporter system was constructed after expanded culture.

In order to ensure that EGFP is correctly inserted in the genome and can be stably expressed, the DNA of the cells is extracted and the target segment is sequenced, which proves that the EGFP gene has been correctly inserted between the coding sequence of the third exon of the kiss1 gene and the 3' non-coding region; at the same time , Western blot (Western blotting) results show that the reporter system inserted into the EGFP coding sequence can correctly express green fluorescent protein, as shown in Figure 4.

3. Establishment of a method for quantitative detection of gene expression levels using flow cytometry

Existing research results have shown that estradiol (E2) up-regulates the expression of the kiss1 gene, while rapamycin (Rapamycin) is an expression inhibitor of the gene, so the above three substances were first used to establish a flow cytometer to quantitatively detect Kiss1 Methods for gene expression levels.

On the first day, cells of the dual-color reporter system with good growth status were selected, and 200,000 cells/well were equally spread into 24-well plates. The next day, after the cells adhered to the wall, the cells were treated with E2 at a total concentration of 100 nM and rapamycin at 70 nM. On the third day, after 24 hours of treatment, use trypsin to digest the cells into a single-cell state, centrifuge to discard the supernatant of the cell digestion solution, use 4% paraformaldehyde to prepare the cell suspension into a cell suspension, and store in a 4°C refrigerator Fix for 30 minutes. Subsequently, flow cytometry analysis was performed, and cells with similar forward scatter and side scatter values, that is, cells with similar cell shapes, were selected for detection. The average red and green fluorescence signal intensity of 10,000 cells with fixed morphology was collected, and the green fluorescence signal was corrected by using the red fluorescence of each treatment group respectively, and the results shown in Figure 5 were obtained: the green fluorescence intensity of cells after estrogen treatment was significantly higher than that of the control group. In order to exclude the influence of the cell state on the analysis results, the red fluorescent signal not regulated by the treatment factors in each group was used to correct the green fluorescent signal, and the significant promotion effect of estrogen on the expression of Kiss1 gene could also be observed.

In order to confirm the reliability of the detection method of the present invention, the currently recognized qPCR and western blot techniques are used to verify the results. When performing qPCR detection, in order to confirm the simultaneous transcription of Kiss1 and EGFP, a cross-Kiss1 coding sequence, P2A sequence and EGFP sequence were designed. detection primers. As shown in Figure 6 and Figure 7, the expression levels of Kiss1 and EGFP increased synchronously after estrogen treatment. From the perspective of detection sensitivity, the flow cytometry used can more sensitively reflect the influence of treatment factors on the regulation of Kiss1 gene expression through the statistical analysis of 10,000 data, and the experiment repeatability is high, which is conducive to the detection of drugs. Statistically significant analysis, while qPCR and western blot cannot achieve similar effects in this respect.

Using the same method, the extremely significant inhibitory effect of rapamycin on Kiss1 gene expression was also detected, as shown in FIG. 8 .

4. High-throughput drug screening

A two-color fluorescent reporter system was used to screen 23 ketone compounds, and the results shown in Figure 9 were obtained. Among the 23 compounds, 9 compounds were detected to significantly affect (p<0.01) the expression of the kiss1 gene, and 2 compounds were verified by qRT-PCR. Such screening work greatly saves the time and money cost of large-scale qPCR for sample preparation, RNA extraction, reverse transcription and real-time quantitative PCR.

5. Microplate reader detects drug-treated two-color fluorescent reporting system

A multifunctional microplate reader is also used to detect the fluorescence intensity. However, for genes with low levels of expression and signals with very small changes, microplate reader detection technology is insufficient. To further illustrate the sensitivity of the flow cytometry analysis method adopted, after the cells were treated with drugs and E2, rapamycin, the fluorescence value in each well was detected with a fluorescent microplate reader, and each sample had 3 repetitions, Take the average value of three wells to obtain the relative fluorescence intensity EGFP/tdtomato, the results are as follows:

Table 1 The influence of compounds detected by multifunctional microplate reader on the expression of kiss1 gene

Differences between compounds were not significant, and differences were not detectable between estrogen, rapamycin, and a negative control that received no treatment.

It can be seen that the use of flow cytometry on the basis of high-throughput, because of the characteristics of analyzing cells one by one, also improves the sensitivity of detecting small differences.

Claims (3)

1. a kind of carry out the method that quantitatively detects of destination gene expression using flow cytometer, it is characterised in that step is：

Step 1：In the cell for expressing target gene to be studied, red fluorescent protein gene is knocked in；In the target gene Reading frame in, knock in green fluorescence protein gene；Red, green fluorescence protein gene is set to be expressed simultaneously with the target gene, But respective albumen is formed independently of one another；Formed it is this can and meanwhile express red, green two kinds of fluorescins cell be used for pair The external factor of influence destination gene expression is screened；

Step 2：External factor enters after the cell that step 1 is formed, and the expression to target gene can produce up-regulation or lower Influence, the influence can be reflected on the expression quantity of green fluorescent protein；The green glimmering of cell is quantitatively detected with flow cytometer The power of optical signal, then uniformed with its red fluorescent value, so as to the external factor to destination gene expression Influence carry out quantitative comparison, quantitatively detect influence destination gene expression external factor.

2. a kind of method quantitatively detected using flow cytometer progress destination gene expression as claimed in claim 1, it is special Levy and be：In the step 1, external factor includes micromolecular compound, microRNA, protein expression vector.

3. a kind of method quantitatively detected using flow cytometer progress destination gene expression as claimed in claim 1 or 2, its It is characterised by：In the step 1, the downstream of target gene coded sequence is inserted into frame using EGFP coded sequence, two The coded sequence for the polypeptide P2A that self cutting can occur in translation is introduced between person, then red, green two kinds of fluorescins can Translated simultaneously but independently of one another, and the upstream and downstream transcription and translation control region of target gene is intactly remained in gene level Domain, EGFP expression quantity is capable of influence of the external factor of loyal reflection application to destination gene expression.