CN113234809A - Method for researching epigenetic stability of pluripotent stem cells - Google Patents
Method for researching epigenetic stability of pluripotent stem cells Download PDFInfo
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Abstract
The invention discloses a research method for epigenetic stability of pluripotent stem cells, which comprises the following steps: taking a mother mouse with 13.5 days of pregnancy, taking out a uterus after the death by a cervical dislocation method, placing the uterus in a culture dish filled with a culture solution, taking out the mouse body in the uterus, taking out an internal embryo, and placing the taken-out embryo in another culture dish filled with the culture solution; by adopting the pregnant mouse, on one hand, the gene sequence of the white mouse in the mouse is similar to that of the human, the similarity of the whole genome and the human is extremely high, and a plurality of diseases which are difficult to cure by the human can be found out in the white mouse, so that the disease curing gene can be found by experiments, on the other hand, the biological significance of the special white mouse for experiments is larger, the cultured mouse almost has no individual difference, whether the physiological individual difference is not mean that the difference in the quality is unknown, but the pure experimental white mouse is mainly selected to reduce the congenital individual difference as much as possible.
Description
Technical Field
The invention belongs to the technical field of stem cell research, and particularly relates to a research method for epigenetic stability of pluripotent stem cells.
Background
Pluripotency (pluripotency) refers to the ability to form more than one type of cell in the body, but is often specific to a particular cell line. Generally a system has several qualitatively different roles, in particular in generative architecture, a part of the embryo that is developing, with several different processes of development, appears to have the ability to form different morphologies; also, the term "cell" refers to a cell or tissue that has a certain morphology and function, and can exhibit various other morphologies and functions due to a change in internal or external conditions. The former is applicable to an unformed embryonic region, for example, the outer embryonic leaf before the formation of the vertebrate primitive gut has pluripotency. The latter case applies to deformations or metamorphoses, for example the pigment layer of an amphibian eye has pluripotency. Further, a multipotent concept in the field of phylogenetics or physiology is used as described above.
Stem cells are a class of cells that have unlimited or immortal self-renewal capacity, capable of producing at least one type of highly differentiated progeny cells.
Phenotype (also called a trait) refers to a trait or characteristic that an organism (or cell) can observe as a result of a specific genotype interacting with the environment. Including the expression of various aspects of individual form, function and the like, such as height, skin color, blood type, enzyme activity, drug tolerance, character and the like. Classical genetics (genetics) refers to changes in gene function due to changes in gene sequence (e.g., gene mutations, etc.) that result in heritable changes in phenotype; epigenetics, on the other hand, refers to heritable changes in gene function without changes in the DNA sequence of the gene, which ultimately results in phenotypic changes.
Therefore, the influencing factors of the epigenetic stability of the pluripotent stem cells are the research targets which are needed at present to define the relationship and action mechanism of the epigenetic stability and the directional differentiation of the pluripotent stem cells.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the existing defects and provide a research method for the epigenetic stability of pluripotent stem cells so as to solve the problem of influence factors of the epigenetic stability of the pluripotent stem cells proposed in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a method for researching epigenetic stability of pluripotent stem cells comprises the following steps:
the method comprises the following steps: taking a mother mouse with 13.5 days of pregnancy, taking out a uterus after the death by a cervical dislocation method, placing the uterus in a culture dish filled with a culture solution, taking out the mouse body in the uterus, taking out an internal embryo, and placing the taken-out embryo in another culture dish filled with the culture solution;
step two: removing heads, limbs, tails and livers of intrauterine mice, cleaning twice by using a cleaning solution, cutting into pieces, placing the cut pieces in another culture dish, sucking out the cleaning solution, adding 3ml of PBS (phosphate buffer solution), 2m of 10.05 percent of pancreatin/EDTA (ethylene diamine tetraacetic acid), adding 5ml of fibroblast culture solution to stop pancreatin digestion after three minutes, 900rpm and 10min, removing the supernatant at the upper end, adding 2-3ml of fibroblast culture solution, resuspending cell precipitates, supplementing the culture solution, placing the culture solution in the culture dish for culture, replacing the culture solution on the next day to obtain primary embryonic fibroblasts, marking the primary embryonic fibroblasts as P0 generation, and then transferring the primary embryonic fibroblasts to P4-P5 generation to be used as feeder cells;
step three: treating with mitomycin when the density of the cells of the P2 generation is about 80%, taking out a part, leaving about 2ml, adding mitomycin to make the concentration about 10 mug/ml, culturing in an incubator for two hours, controlling the oxygen density in the incubator to be 5-21%, taking out the cells after two hours, and freezing for standby treatment;
step four: after the cells subjected to cryopreservation treatment are taken out, the cells are pretreated by using an affinity enrichment method, after the pretreatment is completed, the cells are subjected to bisulfite treatment, PCR amplification is carried out, then DNA purification and recovery are carried out, and then T/A cloning treatment is carried out;
step five: and finally, sequencing the cells subjected to T/last T/A clone treatment in an observation box 1, comparing and analyzing the cells with the original sequence, and recording data.
Preferably, in the second step, after the primary embryonic fibroblasts are transferred to the generation P4-P5, the cells can be used as feeder cells after being treated by mitomycin or by irradiation.
Preferably, in the fourth step, the affinity enrichment pretreatment is to use 5-methylcytosine specific antibody to enrich methylated fragments after breaking and denaturing the genomic DNA by ultrasonic wave, and then separate and purify to obtain methylated DNA fragments.
Preferably, in step four, after bisulfite treatment of the cells, the cytosine (C) in the original DNA sample is converted to uracil (U), and the conversion of 5-methylcytosine to uracil (U) is very low, thereby facilitating detection of uracil (U).
Preferably, in the fourth step, the T/A cloning treatment is to add a template-independent A at the 3 'end of the PCR product by using the Tap enzyme, and the T vector is a vector with a 3' T overhang, so that the PCR product can be rapidly and directly inserted into the Multiple Cloning Site (MCS) of the plasmid vector in one step under the action of the ligase.
Preferably, the T vector is a special plasmid vector for efficiently cloning a PCR product (TA clone), is a linearized vector, and can be directly connected with the PCR product with the A terminal without enzyme digestion.
The utility model provides a research method of epigenetic stability of multipotential stem cell, includes the observation box, observation box one side upper end is provided with the sight glass, observation box one side surface middle part is provided with display and control button respectively, observation window one side surface lower extreme articulates through the hinge has the chamber door, the chamber door surface is provided with the handle, observation box opposite side surface is provided with the radiator-grid, observation box lower extreme four corners all is provided with removes the wheel, it is provided with four, four to remove and all install arresting gear on the wheel.
Preferably, the inside bottom of observation case is provided with the motor case, the inside rotation motor that is provided with of motor case, it is connected with the tray to rotate the transmission of motor upper end, the inside fixedly connected with baffle of observation case, the baffle lower extreme is provided with micro-camera device, an inside side surface of observation case is provided with the ultraviolet ray disinfection lamp.
Compared with the prior art, the invention provides a research method for epigenetic stability of pluripotent stem cells, which has the following beneficial effects:
1. according to the invention, by adopting a mother mouse with 13.5 days of pregnancy, on one hand, the gene sequence of a mouse in the mouse is almost the same as that of a human, the similarity between the whole genome of the mouse and the human is extremely high, and many diseases which are difficult to cure by the human can be found out in the mouse, so that an experiment is carried out to find an illness treatment gene, on the other hand, the biological significance of the special experimental mouse is larger, the cultured mouse almost has no individual difference (physiologically), whether the physiological individual difference means that the physiologism has no difference is unknown or not is unknown, but the pure line experimental mouse is mainly selected to reduce the congenital individual difference as much as possible;
2. according to the invention, the cell is subjected to bisulfite treatment, so that the cytosine (C) in the original DNA sample can be converted into the uracil (U), and the probability of the conversion of the 5-methylcytosine into the uropydine is extremely low, thereby facilitating the detection of the uracil (U).
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention without limiting the invention in which:
FIG. 1 is a system flow diagram of a method for studying epigenetic stability of pluripotent stem cells according to the present invention;
FIG. 2 is a schematic structural diagram of an observation box in the method for studying epigenetic stability of pluripotent stem cells according to the present invention;
FIG. 3 is a cross-sectional view of a viewing box in the method for studying epigenetic stability of pluripotent stem cells according to the present invention;
in the figure: 1. an observation box; 2. a moving wheel; 3. a box door; 4. a handle; 5. a display; 6. a control key; 7. an observation mirror; 8. a heat-dissipating web; 9. a motor case; 10. rotating the motor; 11. a tray; 12. an ultraviolet disinfection lamp; 13. a microscopic imaging device; 14. a separator.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a technical scheme that: a method for researching epigenetic stability of pluripotent stem cells comprises the following steps:
the method comprises the following steps: taking a mother mouse with 13.5 days of pregnancy, taking out a uterus after the death by a cervical dislocation method, placing the uterus in a culture dish filled with a culture solution, taking out the mouse body in the uterus, taking out an internal embryo, and placing the taken-out embryo in another culture dish filled with the culture solution;
step two: removing heads, limbs, tails and livers of intrauterine mice, cleaning twice by using a cleaning solution, cutting into pieces, placing the cut pieces in another culture dish, sucking out the cleaning solution, adding 3ml of PBS (phosphate buffer solution), 2m of 10.05 percent of pancreatin/EDTA (ethylene diamine tetraacetic acid), adding 5ml of fibroblast culture solution to stop pancreatin digestion after three minutes, 900rpm and 10min, removing the supernatant at the upper end, adding 2-3ml of fibroblast culture solution, resuspending cell precipitates, supplementing the culture solution, placing the culture solution in the culture dish for culture, replacing the culture solution on the next day to obtain primary embryonic fibroblasts, marking the primary embryonic fibroblasts as P0 generation, and then transferring the primary embryonic fibroblasts to P4-P5 generation to be used as feeder cells;
step three: treating with mitomycin when the density of the cells of the P2 generation is about 80%, taking out a part, leaving about 2ml, adding mitomycin to make the concentration about 10 mug/ml, culturing in an incubator for two hours, controlling the oxygen density in the incubator to be 5-21%, taking out the cells after two hours, and freezing for standby treatment;
step four: after the cells subjected to cryopreservation treatment are taken out, the cells are pretreated by using an affinity enrichment method, after the pretreatment is completed, the cells are subjected to bisulfite treatment, PCR amplification is carried out, then DNA purification and recovery are carried out, and then T/A cloning treatment is carried out;
step five: and finally, sequencing the cells subjected to T/last T/A clone treatment in an observation box 1, comparing and analyzing the cells with the original sequence, and recording data.
In the second step, after the primary embryonic fibroblasts are transferred to the P4-P5 generation, the cells can be used as feeder cells after being treated by mitomycin or by irradiation.
In the fourth step, the pretreatment of the affinity enrichment method is to use 5-methylcytosine specific antibody to enrich methylated fragments after the genomic DNA is broken by ultrasonic waves and denatured, and then separate and purify to obtain the methylated DNA fragments.
In step four, after bisulfite treatment of the cells, cytosine (C) in the original DNA sample can be converted to uracil (U), and the conversion of 5-methylcytosine to uracil (U) is very low, thereby facilitating detection of uracil (U).
In the fourth step, the T/A cloning treatment is to utilize the Tap enzyme to add a template-independent A at the 3 'end of the PCR product, and the T vector is a vector with a 3' T overhang, and the PCR product can be rapidly and directly inserted into the Multiple Cloning Sites (MCS) of the plasmid vector in one step under the action of the ligase.
The T vector is a special plasmid vector for efficiently cloning a PCR product (TA cloning), is a linearized vector, and can be directly connected with the PCR product with the A terminal without enzyme digestion.
The method for researching the epigenetic stability of the pluripotent stem cells comprises an observation box 1, wherein an observation mirror 7 is arranged at the upper end of one side of the observation box 1, a display 5 and a control button 6 are respectively arranged at the middle part of the surface of one side of the observation box 1, a box door 3 is hinged to the lower end of the surface of one side of the observation window 1 through a hinge, a handle 4 is arranged on the surface of the box door 3, a heat dissipation net 8 is arranged on the surface of the other side of the observation box 1, four moving wheels 2 are arranged at four corners of the lower end of the observation box 1, four moving wheels 2 are provided, and a braking device is arranged on each of the four moving wheels 2.
The inside bottom of observation box 1 is provided with motor case 9, and motor case 9 is inside to be provided with and to rotate motor 10, rotates motor 10 upper end transmission and is connected with tray 11, and the inside fixedly connected with baffle 14 of observation box 1, the baffle 14 lower extreme are provided with micro-camera device 13, and 1 inside side surface of observation box is provided with ultraviolet ray disinfection lamp 12.
Example one
A method for researching epigenetic stability of pluripotent stem cells comprises the following steps:
the method comprises the following steps: taking a mother mouse with 13.5 days of pregnancy, taking out a uterus after the death by a cervical dislocation method, placing the uterus in a culture dish filled with a culture solution, taking out the mouse body in the uterus, taking out an internal embryo, and placing the taken-out embryo in another culture dish filled with the culture solution;
step two: removing heads, limbs, tails and livers of intrauterine mice, cleaning twice by using a cleaning solution, cutting into pieces, placing the cut pieces in another culture dish, sucking out the cleaning solution, adding 3ml of PBS (phosphate buffer solution), 2m of 10.05 percent of pancreatin/EDTA (ethylene diamine tetraacetic acid), adding 5ml of fibroblast culture solution to stop pancreatin digestion after three minutes, 900rpm and 10min, removing the supernatant at the upper end, adding 2-3ml of fibroblast culture solution, resuspending cell precipitates, supplementing the culture solution, placing the culture solution in the culture dish for culture, replacing the culture solution on the next day to obtain primary embryonic fibroblasts, marking the primary embryonic fibroblasts as P0 generation, and then transferring the primary embryonic fibroblasts to P4-P5 generation to be used as feeder cells;
step three: treating with mitomycin when the density of the cells of the P2 generation is about 80%, taking out a part, leaving about 2ml, adding mitomycin to make the concentration about 10 mug/ml, culturing in an incubator for two hours, controlling the oxygen density in the incubator to be 5%, taking out the cells after two hours, and freezing for standby treatment;
step four: after the cells subjected to cryopreservation treatment are taken out, the cells are pretreated by using an affinity enrichment method, after the pretreatment is completed, the cells are subjected to bisulfite treatment, PCR amplification is carried out, then DNA purification and recovery are carried out, and then T/A cloning treatment is carried out;
step five: and finally, sequencing the cells subjected to T/last T/A clone treatment in an observation box 1, comparing and analyzing the cells with the original sequence, and recording data.
In the second step, after the primary embryonic fibroblasts are transferred to the P4-P5 generation, the cells can be used as feeder cells after being treated by mitomycin or by irradiation.
In the fourth step, the pretreatment of the affinity enrichment method is to use 5-methylcytosine specific antibody to enrich methylated fragments after the genomic DNA is broken by ultrasonic waves and denatured, and then separate and purify to obtain the methylated DNA fragments.
In step four, after bisulfite treatment of the cells, cytosine (C) in the original DNA sample can be converted to uracil (U), and the conversion of 5-methylcytosine to uracil (U) is very low, thereby facilitating detection of uracil (U).
In the fourth step, the T/A cloning treatment is to utilize the Tap enzyme to add a template-independent A at the 3 'end of the PCR product, and the T vector is a vector with a 3' T overhang, and the PCR product can be rapidly and directly inserted into the Multiple Cloning Sites (MCS) of the plasmid vector in one step under the action of the ligase.
The T vector is a special plasmid vector for efficiently cloning a PCR product (TA cloning), is a linearized vector, and can be directly connected with the PCR product with the A terminal without enzyme digestion.
The method for researching the epigenetic stability of the pluripotent stem cells comprises an observation box 1, wherein an observation mirror 7 is arranged at the upper end of one side of the observation box 1, a display 5 and a control button 6 are respectively arranged at the middle part of the surface of one side of the observation box 1, a box door 3 is hinged to the lower end of the surface of one side of the observation window 1 through a hinge, a handle 4 is arranged on the surface of the box door 3, a heat dissipation net 8 is arranged on the surface of the other side of the observation box 1, four moving wheels 2 are arranged at four corners of the lower end of the observation box 1, four moving wheels 2 are provided, and a braking device is arranged on each of the four moving wheels 2.
The inside bottom of observation box 1 is provided with motor case 9, and motor case 9 is inside to be provided with and to rotate motor 10, rotates motor 10 upper end transmission and is connected with tray 11, and the inside fixedly connected with baffle 14 of observation box 1, the baffle 14 lower extreme are provided with micro-camera device 13, and 1 inside side surface of observation box is provided with ultraviolet ray disinfection lamp 12.
Example two
A method for researching epigenetic stability of pluripotent stem cells comprises the following steps:
the method comprises the following steps: taking a mother mouse with 13.5 days of pregnancy, taking out a uterus after the death by a cervical dislocation method, placing the uterus in a culture dish filled with a culture solution, taking out the mouse body in the uterus, taking out an internal embryo, and placing the taken-out embryo in another culture dish filled with the culture solution;
step two: removing heads, limbs, tails and livers of intrauterine mice, cleaning twice by using a cleaning solution, cutting into pieces, placing the cut pieces in another culture dish, sucking out the cleaning solution, adding 3ml of PBS (phosphate buffer solution), 2m of 10.05 percent of pancreatin/EDTA (ethylene diamine tetraacetic acid), adding 5ml of fibroblast culture solution to stop pancreatin digestion after three minutes, 900rpm and 10min, removing the supernatant at the upper end, adding 2-3ml of fibroblast culture solution, resuspending cell precipitates, supplementing the culture solution, placing the culture solution in the culture dish for culture, replacing the culture solution on the next day to obtain primary embryonic fibroblasts, marking the primary embryonic fibroblasts as P0 generation, and then transferring the primary embryonic fibroblasts to P4-P5 generation to be used as feeder cells;
step three: treating with mitomycin when the density of the cells of the P2 generation is about 80%, taking out a part, leaving about 2ml, adding mitomycin to make the concentration about 10 mug/ml, culturing in an incubator for two hours, controlling the oxygen density in the incubator to be 8%, taking out the cells after two hours, and freezing for standby treatment;
step four: after the cells subjected to cryopreservation treatment are taken out, the cells are pretreated by using an affinity enrichment method, after the pretreatment is completed, the cells are subjected to bisulfite treatment, PCR amplification is carried out, then DNA purification and recovery are carried out, and then T/A cloning treatment is carried out;
step five: and finally, sequencing the cells subjected to T/last T/A clone treatment in an observation box 1, comparing and analyzing the cells with the original sequence, and recording data.
In the second step, after the primary embryonic fibroblasts are transferred to the P4-P5 generation, the cells can be used as feeder cells after being treated by mitomycin or by irradiation.
In the fourth step, the pretreatment of the affinity enrichment method is to use 5-methylcytosine specific antibody to enrich methylated fragments after the genomic DNA is broken by ultrasonic waves and denatured, and then separate and purify to obtain the methylated DNA fragments.
In step four, after bisulfite treatment of the cells, cytosine (C) in the original DNA sample can be converted to uracil (U), and the conversion of 5-methylcytosine to uracil (U) is very low, thereby facilitating detection of uracil (U).
In the fourth step, the T/A cloning treatment is to utilize the Tap enzyme to add a template-independent A at the 3 'end of the PCR product, and the T vector is a vector with a 3' T overhang, and the PCR product can be rapidly and directly inserted into the Multiple Cloning Sites (MCS) of the plasmid vector in one step under the action of the ligase.
The T vector is a special plasmid vector for efficiently cloning a PCR product (TA cloning), is a linearized vector, and can be directly connected with the PCR product with the A terminal without enzyme digestion.
The method for researching the epigenetic stability of the pluripotent stem cells comprises an observation box 1, wherein an observation mirror 7 is arranged at the upper end of one side of the observation box 1, a display 5 and a control button 6 are respectively arranged at the middle part of the surface of one side of the observation box 1, a box door 3 is hinged to the lower end of the surface of one side of the observation window 1 through a hinge, a handle 4 is arranged on the surface of the box door 3, a heat dissipation net 8 is arranged on the surface of the other side of the observation box 1, four moving wheels 2 are arranged at four corners of the lower end of the observation box 1, four moving wheels 2 are provided, and a braking device is arranged on each of the four moving wheels 2.
The inside bottom of observation box 1 is provided with motor case 9, and motor case 9 is inside to be provided with and to rotate motor 10, rotates motor 10 upper end transmission and is connected with tray 11, and the inside fixedly connected with baffle 14 of observation box 1, the baffle 14 lower extreme are provided with micro-camera device 13, and 1 inside side surface of observation box is provided with ultraviolet ray disinfection lamp 12.
EXAMPLE III
A method for researching epigenetic stability of pluripotent stem cells comprises the following steps:
the method comprises the following steps: taking a mother mouse with 13.5 days of pregnancy, taking out a uterus after the death by a cervical dislocation method, placing the uterus in a culture dish filled with a culture solution, taking out the mouse body in the uterus, taking out an internal embryo, and placing the taken-out embryo in another culture dish filled with the culture solution;
step two: removing heads, limbs, tails and livers of intrauterine mice, cleaning twice by using a cleaning solution, cutting into pieces, placing the cut pieces in another culture dish, sucking out the cleaning solution, adding 3ml of PBS (phosphate buffer solution), 2m of 10.05 percent of pancreatin/EDTA (ethylene diamine tetraacetic acid), adding 5ml of fibroblast culture solution to stop pancreatin digestion after three minutes, 900rpm and 10min, removing the supernatant at the upper end, adding 2-3ml of fibroblast culture solution, resuspending cell precipitates, supplementing the culture solution, placing the culture solution in the culture dish for culture, replacing the culture solution on the next day to obtain primary embryonic fibroblasts, marking the primary embryonic fibroblasts as P0 generation, and then transferring the primary embryonic fibroblasts to P4-P5 generation to be used as feeder cells;
step three: treating with mitomycin when the density of the cells of the P2 generation is about 80%, taking out a part, leaving about 2ml, adding mitomycin to make the concentration about 10 mug/ml, culturing in an incubator for two hours, controlling the oxygen density in the incubator to be 11%, taking out the cells after two hours, and freezing for standby treatment;
step four: after the cells subjected to cryopreservation treatment are taken out, the cells are pretreated by using an affinity enrichment method, after the pretreatment is completed, the cells are subjected to bisulfite treatment, PCR amplification is carried out, then DNA purification and recovery are carried out, and then T/A cloning treatment is carried out;
step five: and finally, sequencing the cells subjected to T/last T/A clone treatment in an observation box 1, comparing and analyzing the cells with the original sequence, and recording data.
In the second step, after the primary embryonic fibroblasts are transferred to the P4-P5 generation, the cells can be used as feeder cells after being treated by mitomycin or by irradiation.
In the fourth step, the pretreatment of the affinity enrichment method is to use 5-methylcytosine specific antibody to enrich methylated fragments after the genomic DNA is broken by ultrasonic waves and denatured, and then separate and purify to obtain the methylated DNA fragments.
In step four, after bisulfite treatment of the cells, cytosine (C) in the original DNA sample can be converted to uracil (U), and the conversion of 5-methylcytosine to uracil (U) is very low, thereby facilitating detection of uracil (U).
In the fourth step, the T/A cloning treatment is to utilize the Tap enzyme to add a template-independent A at the 3 'end of the PCR product, and the T vector is a vector with a 3' T overhang, and the PCR product can be rapidly and directly inserted into the Multiple Cloning Sites (MCS) of the plasmid vector in one step under the action of the ligase.
The T vector is a special plasmid vector for efficiently cloning a PCR product (TA cloning), is a linearized vector, and can be directly connected with the PCR product with the A terminal without enzyme digestion.
The method for researching the epigenetic stability of the pluripotent stem cells comprises an observation box 1, wherein an observation mirror 7 is arranged at the upper end of one side of the observation box 1, a display 5 and a control button 6 are respectively arranged at the middle part of the surface of one side of the observation box 1, a box door 3 is hinged to the lower end of the surface of one side of the observation window 1 through a hinge, a handle 4 is arranged on the surface of the box door 3, a heat dissipation net 8 is arranged on the surface of the other side of the observation box 1, four moving wheels 2 are arranged at four corners of the lower end of the observation box 1, four moving wheels 2 are provided, and a braking device is arranged on each of the four moving wheels 2.
The inside bottom of observation box 1 is provided with motor case 9, and motor case 9 is inside to be provided with and to rotate motor 10, rotates motor 10 upper end transmission and is connected with tray 11, and the inside fixedly connected with baffle 14 of observation box 1, the baffle 14 lower extreme are provided with micro-camera device 13, and 1 inside side surface of observation box is provided with ultraviolet ray disinfection lamp 12.
Example four
A method for researching epigenetic stability of pluripotent stem cells comprises the following steps:
the method comprises the following steps: taking a mother mouse with 13.5 days of pregnancy, taking out a uterus after the death by a cervical dislocation method, placing the uterus in a culture dish filled with a culture solution, taking out the mouse body in the uterus, taking out an internal embryo, and placing the taken-out embryo in another culture dish filled with the culture solution;
step two: removing heads, limbs, tails and livers of intrauterine mice, cleaning twice by using a cleaning solution, cutting into pieces, placing the cut pieces in another culture dish, sucking out the cleaning solution, adding 3ml of PBS (phosphate buffer solution), 2m of 10.05 percent of pancreatin/EDTA (ethylene diamine tetraacetic acid), adding 5ml of fibroblast culture solution to stop pancreatin digestion after three minutes, 900rpm and 10min, removing the supernatant at the upper end, adding 2-3ml of fibroblast culture solution, resuspending cell precipitates, supplementing the culture solution, placing the culture solution in the culture dish for culture, replacing the culture solution on the next day to obtain primary embryonic fibroblasts, marking the primary embryonic fibroblasts as P0 generation, and then transferring the primary embryonic fibroblasts to P4-P5 generation to be used as feeder cells;
step three: treating with mitomycin when the density of the cells of the P2 generation is about 80%, taking out a part, leaving about 2ml, adding mitomycin to make the concentration about 10 mug/ml, culturing in an incubator for two hours, controlling the oxygen density in the incubator to be 15%, taking out the cells after two hours, and freezing for standby treatment;
step four: after the cells subjected to cryopreservation treatment are taken out, the cells are pretreated by using an affinity enrichment method, after the pretreatment is completed, the cells are subjected to bisulfite treatment, PCR amplification is carried out, then DNA purification and recovery are carried out, and then T/A cloning treatment is carried out;
step five: and finally, sequencing the cells subjected to T/last T/A clone treatment in an observation box 1, comparing and analyzing the cells with the original sequence, and recording data.
In the second step, after the primary embryonic fibroblasts are transferred to the P4-P5 generation, the cells can be used as feeder cells after being treated by mitomycin or by irradiation.
In the fourth step, the pretreatment of the affinity enrichment method is to use 5-methylcytosine specific antibody to enrich methylated fragments after the genomic DNA is broken by ultrasonic waves and denatured, and then separate and purify to obtain the methylated DNA fragments.
In step four, after bisulfite treatment of the cells, cytosine (C) in the original DNA sample can be converted to uracil (U), and the conversion of 5-methylcytosine to uracil (U) is very low, thereby facilitating detection of uracil (U).
In the fourth step, the T/A cloning treatment is to utilize the Tap enzyme to add a template-independent A at the 3 'end of the PCR product, and the T vector is a vector with a 3' T overhang, and the PCR product can be rapidly and directly inserted into the Multiple Cloning Sites (MCS) of the plasmid vector in one step under the action of the ligase.
The T vector is a special plasmid vector for efficiently cloning a PCR product (TA cloning), is a linearized vector, and can be directly connected with the PCR product with the A terminal without enzyme digestion.
The method for researching the epigenetic stability of the pluripotent stem cells comprises an observation box 1, wherein an observation mirror 7 is arranged at the upper end of one side of the observation box 1, a display 5 and a control button 6 are respectively arranged at the middle part of the surface of one side of the observation box 1, a box door 3 is hinged to the lower end of the surface of one side of the observation window 1 through a hinge, a handle 4 is arranged on the surface of the box door 3, a heat dissipation net 8 is arranged on the surface of the other side of the observation box 1, four moving wheels 2 are arranged at four corners of the lower end of the observation box 1, four moving wheels 2 are provided, and a braking device is arranged on each of the four moving wheels 2.
The inside bottom of observation box 1 is provided with motor case 9, and motor case 9 is inside to be provided with and to rotate motor 10, rotates motor 10 upper end transmission and is connected with tray 11, and the inside fixedly connected with baffle 14 of observation box 1, the baffle 14 lower extreme are provided with micro-camera device 13, and 1 inside side surface of observation box is provided with ultraviolet ray disinfection lamp 12.
EXAMPLE five
A method for researching epigenetic stability of pluripotent stem cells comprises the following steps:
the method comprises the following steps: taking a mother mouse with 13.5 days of pregnancy, taking out a uterus after the death by a cervical dislocation method, placing the uterus in a culture dish filled with a culture solution, taking out the mouse body in the uterus, taking out an internal embryo, and placing the taken-out embryo in another culture dish filled with the culture solution;
step two: removing heads, limbs, tails and livers of intrauterine mice, cleaning twice by using a cleaning solution, cutting into pieces, placing the cut pieces in another culture dish, sucking out the cleaning solution, adding 3ml of PBS (phosphate buffer solution), 2m of 10.05 percent of pancreatin/EDTA (ethylene diamine tetraacetic acid), adding 5ml of fibroblast culture solution to stop pancreatin digestion after three minutes, 900rpm and 10min, removing the supernatant at the upper end, adding 2-3ml of fibroblast culture solution, resuspending cell precipitates, supplementing the culture solution, placing the culture solution in the culture dish for culture, replacing the culture solution on the next day to obtain primary embryonic fibroblasts, marking the primary embryonic fibroblasts as P0 generation, and then transferring the primary embryonic fibroblasts to P4-P5 generation to be used as feeder cells;
step three: treating with mitomycin when the density of the cells of the P2 generation is about 80%, taking out a part, leaving about 2ml, adding mitomycin to make the concentration about 10 mug/ml, culturing in an incubator for two hours, controlling the oxygen density in the incubator to be 18%, taking out the cells after two hours, and freezing for standby treatment;
step four: after the cells subjected to cryopreservation treatment are taken out, the cells are pretreated by using an affinity enrichment method, after the pretreatment is completed, the cells are subjected to bisulfite treatment, PCR amplification is carried out, then DNA purification and recovery are carried out, and then T/A cloning treatment is carried out;
step five: and finally, sequencing the cells subjected to T/last T/A clone treatment in an observation box 1, comparing and analyzing the cells with the original sequence, and recording data.
In the second step, after the primary embryonic fibroblasts are transferred to the P4-P5 generation, the cells can be used as feeder cells after being treated by mitomycin or by irradiation.
In the fourth step, the pretreatment of the affinity enrichment method is to use 5-methylcytosine specific antibody to enrich methylated fragments after the genomic DNA is broken by ultrasonic waves and denatured, and then separate and purify to obtain the methylated DNA fragments.
In step four, after bisulfite treatment of the cells, cytosine (C) in the original DNA sample can be converted to uracil (U), and the conversion of 5-methylcytosine to uracil (U) is very low, thereby facilitating detection of uracil (U).
In the fourth step, the T/A cloning treatment is to utilize the Tap enzyme to add a template-independent A at the 3 'end of the PCR product, and the T vector is a vector with a 3' T overhang, and the PCR product can be rapidly and directly inserted into the Multiple Cloning Sites (MCS) of the plasmid vector in one step under the action of the ligase.
The T vector is a special plasmid vector for efficiently cloning a PCR product (TA cloning), is a linearized vector, and can be directly connected with the PCR product with the A terminal without enzyme digestion.
The method for researching the epigenetic stability of the pluripotent stem cells comprises an observation box 1, wherein an observation mirror 7 is arranged at the upper end of one side of the observation box 1, a display 5 and a control button 6 are respectively arranged at the middle part of the surface of one side of the observation box 1, a box door 3 is hinged to the lower end of the surface of one side of the observation window 1 through a hinge, a handle 4 is arranged on the surface of the box door 3, a heat dissipation net 8 is arranged on the surface of the other side of the observation box 1, four moving wheels 2 are arranged at four corners of the lower end of the observation box 1, four moving wheels 2 are provided, and a braking device is arranged on each of the four moving wheels 2.
The inside bottom of observation box 1 is provided with motor case 9, and motor case 9 is inside to be provided with and to rotate motor 10, rotates motor 10 upper end transmission and is connected with tray 11, and the inside fixedly connected with baffle 14 of observation box 1, the baffle 14 lower extreme are provided with micro-camera device 13, and 1 inside side surface of observation box is provided with ultraviolet ray disinfection lamp 12.
EXAMPLE six
A method for researching epigenetic stability of pluripotent stem cells comprises the following steps:
the method comprises the following steps: taking a mother mouse with 13.5 days of pregnancy, taking out a uterus after the death by a cervical dislocation method, placing the uterus in a culture dish filled with a culture solution, taking out the mouse body in the uterus, taking out an internal embryo, and placing the taken-out embryo in another culture dish filled with the culture solution;
step two: removing heads, limbs, tails and livers of intrauterine mice, cleaning twice by using a cleaning solution, cutting into pieces, placing the cut pieces in another culture dish, sucking out the cleaning solution, adding 3ml of PBS (phosphate buffer solution), 2m of 10.05 percent of pancreatin/EDTA (ethylene diamine tetraacetic acid), adding 5ml of fibroblast culture solution to stop pancreatin digestion after three minutes, 900rpm and 10min, removing the supernatant at the upper end, adding 2-3ml of fibroblast culture solution, resuspending cell precipitates, supplementing the culture solution, placing the culture solution in the culture dish for culture, replacing the culture solution on the next day to obtain primary embryonic fibroblasts, marking the primary embryonic fibroblasts as P0 generation, and then transferring the primary embryonic fibroblasts to P4-P5 generation to be used as feeder cells;
step three: treating with mitomycin when the density of the cells of the P2 generation is about 80%, taking out a part, leaving about 2ml, adding mitomycin to make the concentration about 10 mug/ml, culturing in an incubator for two hours, controlling the oxygen density in the incubator to be 21%, taking out the cells after two hours, and freezing for standby treatment;
step four: after the cells subjected to cryopreservation treatment are taken out, the cells are pretreated by using an affinity enrichment method, after the pretreatment is completed, the cells are subjected to bisulfite treatment, PCR amplification is carried out, then DNA purification and recovery are carried out, and then T/A cloning treatment is carried out;
step five: and finally, sequencing the cells subjected to T/last T/A clone treatment in an observation box 1, comparing and analyzing the cells with the original sequence, and recording data.
In the second step, after the primary embryonic fibroblasts are transferred to the P4-P5 generation, the cells can be used as feeder cells after being treated by mitomycin or by irradiation.
In the fourth step, the pretreatment of the affinity enrichment method is to use 5-methylcytosine specific antibody to enrich methylated fragments after the genomic DNA is broken by ultrasonic waves and denatured, and then separate and purify to obtain the methylated DNA fragments.
In step four, after bisulfite treatment of the cells, cytosine (C) in the original DNA sample can be converted to uracil (U), and the conversion of 5-methylcytosine to uracil (U) is very low, thereby facilitating detection of uracil (U).
In the fourth step, the T/A cloning treatment is to utilize the Tap enzyme to add a template-independent A at the 3 'end of the PCR product, and the T vector is a vector with a 3' T overhang, and the PCR product can be rapidly and directly inserted into the Multiple Cloning Sites (MCS) of the plasmid vector in one step under the action of the ligase.
The T vector is a special plasmid vector for efficiently cloning a PCR product (TA cloning), is a linearized vector, and can be directly connected with the PCR product with the A terminal without enzyme digestion.
The working principle and the using process of the invention are as follows: when in use, a mother mouse with 13.5 days of pregnancy is taken, after the neck breaking method is used for death, the uterus is taken out and is placed in a culture dish filled with culture solution, the mouse body in the uterus is taken out, the embryo in the uterus is taken out, the taken embryo is placed in another culture dish filled with culture solution, the head, the limbs, the tail and the liver of the mouse body in the uterus are removed, then the mouse body is cleaned twice by cleaning solution and is cut into pieces, the cleaning solution is placed in another culture dish, the cleaning solution is sucked out and is added with 3ml of PBS, 2m 10.05 percent of pancreatin/EDTA, after three minutes, 5ml of fibroblast culture solution is added to stop the pancreatin digestion, 900rpm and 10min are carried out, the clear solution at the upper end is removed, 2 to 3ml of fibroblast culture solution is added, the cell sediment is resuspended, the culture solution is supplemented and is placed in the culture dish for culture, the culture solution is replaced the next day, the primary fibroblast is obtained and is marked as P0, subculturing to P4-P5 generation, using as feeder layer cell, treating with mitomycin when density of P2 generation cell is about 80%, taking out part, leaving about 2ml, adding mitomycin to make its concentration be about 10 mug/ml, then placing into incubator to culture for two hours, controlling oxygen density in incubator to be 5-20%, taking out cell after two hours, freezing for standby treatment, taking out cell after freezing treatment, pretreating cell by affinity enrichment method, after pretreatment, carrying out bisulfite treatment, PCR amplification, DNA purification and recovery, then carrying out T/A clone treatment, finally sequencing T/A clone treated cell in observation box 1, comparing and analyzing with original sequence, the data is recorded.
In conclusion, the expression level of the embryo cell imprinting gene can be changed by controlling the oxygen concentration in the incubator, namely controlling the oxygen concentration in the embryo growth environment, and when the oxygen concentration is about 18%, the expression level of the embryo cell imprinting gene is influenced the least by the oxygen concentration, and the difference multiple of the two is about 1.60E + 00.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A method for researching epigenetic stability of pluripotent stem cells is characterized in that: the method comprises the following steps:
the method comprises the following steps: taking a mother mouse with 13.5 days of pregnancy, taking out a uterus after the death by a cervical dislocation method, placing the uterus in a culture dish filled with a culture solution, taking out the mouse body in the uterus, taking out an internal embryo, and placing the taken-out embryo in another culture dish filled with the culture solution;
step two: removing heads, limbs, tails and livers of intrauterine mice, cleaning twice by using a cleaning solution, cutting into pieces, placing the cut pieces in another culture dish, sucking out the cleaning solution, adding 3ml of PBS and 2m of 10.05% pancreatin/EDTA, adding 5ml of fibroblast culture solution to stop pancreatin digestion after three minutes, 900rpm for 10min, removing the supernatant at the upper end, adding 2-3ml of fibroblast culture solution, resuspending cell precipitates, supplementing the culture solution, placing the culture solution in the culture dish for culture, replacing the culture solution on the next day to obtain primary embryonic fibroblasts, marking the primary embryonic fibroblasts as P0 generation, and transferring the primary embryonic fibroblasts to P4-P5 generation to be used as feeder cells;
step three: treating with mitomycin when the density of the cells of the P2 generation is about 80%, taking out a part, leaving about 2ml, adding mitomycin to make the concentration about 10 mug/ml, culturing in an incubator for two hours, controlling the oxygen density in the incubator to be 5-21%, taking out the cells after two hours, and freezing for standby treatment;
step four: after the cells subjected to cryopreservation treatment are taken out, the cells are pretreated by using an affinity enrichment method, after the pretreatment is completed, the cells are subjected to bisulfite treatment, PCR amplification is carried out, then DNA purification and recovery are carried out, and then T/A cloning treatment is carried out;
step five: and finally, sequencing the cells subjected to T/last T/A clone treatment in an observation box 1, comparing and analyzing the cells with the original sequence, and recording data.
2. The method of claim 1, wherein the method comprises the steps of: in the second step, after the primary embryonic fibroblasts are transferred to the generation P4-P5, the cells can be used as feeder cells after being treated by mitomycin or by irradiation.
3. The method of claim 1, wherein the method comprises the steps of: in the fourth step, the pretreatment of the affinity enrichment method is to use 5-methylcytosine specific antibody to enrich methylated fragments after the genomic DNA is broken and denatured by ultrasonic waves, and then separate and purify to obtain the methylated DNA fragments.
4. The method of claim 1, wherein the method comprises the steps of: in the fourth step, after the cells are treated by bisulfite, the cytidines (C) in the original DNA sample can be converted into the uracils (U), and the probability of the conversion of the 5-methylcytosines into the uracils is extremely low, so that the uracils (U) can be conveniently detected.
5. The method of claim 1, wherein the method comprises the steps of: in the fourth step, the T/A cloning treatment is to add a template-independent A at the 3 'end of the PCR product by using the Tap enzyme, and the T vector is a vector with a 3' T overhang, so that the PCR product can be rapidly and directly inserted into the Multiple Cloning Sites (MCS) of the plasmid vector in one step under the action of the ligase.
6. The method of claim 5, wherein the method comprises the steps of: the T vector is a special plasmid vector for efficiently cloning a PCR product (TA clone), is a linearized vector, and can be directly connected with the PCR product with the A terminal without enzyme digestion.
7. A method for researching epigenetic stability of pluripotent stem cells comprises an observation box (1), and is characterized in that: observation box (1) one side upper end is provided with sight glass (7), observation box (1) side surface middle part is provided with display (5) and control button (6) respectively, observation box (1) side surface lower extreme articulates through the hinge has chamber door (3), chamber door (3) surface is provided with handle (4), observation box (1) opposite side surface is provided with radiator-grid (8), observation box (1) lower extreme four corners all is provided with removes wheel (2), it is provided with four, four to remove wheel (2) remove and all install arresting gear on taking turns (2).
8. The method of claim 7, wherein the method comprises the steps of: the utility model discloses an ultraviolet disinfection lamp, including observation case (1), motor case (9) are provided with to inside bottom of observation case (1), motor case (9) inside is provided with rotates motor (10), it is connected with tray (11) to rotate motor (10) upper end transmission, the inside fixedly connected with baffle (14) of observation case (1), baffle (14) lower extreme is provided with micro-camera device (13), an inside side surface of observation case (1) is provided with ultraviolet ray disinfection lamp (12).
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