CN105766795A - New function of genetically engineered mouse for improving Foxb2 expression level - Google Patents
New function of genetically engineered mouse for improving Foxb2 expression level Download PDFInfo
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- CN105766795A CN105766795A CN201610162649.2A CN201610162649A CN105766795A CN 105766795 A CN105766795 A CN 105766795A CN 201610162649 A CN201610162649 A CN 201610162649A CN 105766795 A CN105766795 A CN 105766795A
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- foxb2
- genetically engineered
- dnmt1
- mus
- new function
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- 230000014509 gene expression Effects 0.000 title claims abstract description 16
- 241000699666 Mus <mouse, genus> Species 0.000 claims abstract description 24
- 108091023040 Transcription factor Proteins 0.000 claims abstract description 4
- 102000040945 Transcription factor Human genes 0.000 claims abstract description 4
- 230000008859 change Effects 0.000 claims abstract description 3
- 238000010353 genetic engineering Methods 0.000 claims description 14
- 238000011746 C57BL/6J (JAX™ mouse strain) Methods 0.000 claims description 6
- 101150007297 Dnmt1 gene Proteins 0.000 claims description 3
- 238000003209 gene knockout Methods 0.000 claims 1
- 108010009540 DNA (Cytosine-5-)-Methyltransferase 1 Proteins 0.000 abstract description 10
- 102100036279 DNA (cytosine-5)-methyltransferase 1 Human genes 0.000 abstract description 10
- 108090000623 proteins and genes Proteins 0.000 abstract description 10
- 102000004169 proteins and genes Human genes 0.000 abstract description 8
- 238000000034 method Methods 0.000 abstract description 6
- 108020004414 DNA Proteins 0.000 abstract description 3
- 230000007067 DNA methylation Effects 0.000 abstract description 3
- 230000024245 cell differentiation Effects 0.000 abstract description 3
- 238000000605 extraction Methods 0.000 abstract description 3
- 230000008569 process Effects 0.000 abstract description 3
- 241000699670 Mus sp. Species 0.000 abstract description 2
- 230000002159 abnormal effect Effects 0.000 abstract description 2
- 230000010261 cell growth Effects 0.000 abstract description 2
- 108060004795 Methyltransferase Proteins 0.000 abstract 1
- 102000016397 Methyltransferase Human genes 0.000 abstract 1
- 101100013368 Mus musculus Foxb2 gene Proteins 0.000 abstract 1
- 230000033228 biological regulation Effects 0.000 abstract 1
- 210000003710 cerebral cortex Anatomy 0.000 abstract 1
- 230000013020 embryo development Effects 0.000 abstract 1
- 230000006718 epigenetic regulation Effects 0.000 abstract 1
- 238000010195 expression analysis Methods 0.000 abstract 1
- 238000011813 knockout mouse model Methods 0.000 abstract 1
- 230000011987 methylation Effects 0.000 abstract 1
- 238000007069 methylation reaction Methods 0.000 abstract 1
- 230000007472 neurodevelopment Effects 0.000 abstract 1
- 230000001105 regulatory effect Effects 0.000 description 5
- 210000004556 brain Anatomy 0.000 description 4
- 238000004140 cleaning Methods 0.000 description 4
- 230000001276 controlling effect Effects 0.000 description 4
- 210000005257 cortical tissue Anatomy 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 108090000992 Transferases Proteins 0.000 description 2
- 102000004357 Transferases Human genes 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 238000012822 chemical development Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000002331 protein detection Methods 0.000 description 2
- 238000000751 protein extraction Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000011830 transgenic mouse model Methods 0.000 description 2
- 108010077544 Chromatin Proteins 0.000 description 1
- 230000030933 DNA methylation on cytosine Effects 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 108091027757 Deoxyribozyme Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000289669 Erinaceus europaeus Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 230000008175 fetal development Effects 0.000 description 1
- 235000003869 genetically modified organism Nutrition 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000008271 nervous system development Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
DNMT1 is a DNA methyltransferase and is mainly used for catalyzing a CpG methylation process of DNA. The invention discloses a new function of a genetically engineered mouse obtained through knockout of DNMT1. Extraction and protein expression analysis performed on cerebral cortex of the genetically engineered mouse show that compared with normal mice, the expression quantity of a Foxb2 gene of the genetically engineered mouse is remarkably increased. Foxb2 protein as a transcription factor is believed to have important functions in aspects of organism embryogenesis, cell growth and differentiation as well as cycle extension. Therefore, the change indicates that Foxb2 is subjected to the epigenetic regulation in a DNA methylation form, the knockout mouse possibly has obviously abnormal performance in the aspects of cell differentiation and the like, and the key regulation function of DNMT1 on neurodevelopment is proved.
Description
Technical field
The application belongs to the technical field of genetically modified organism New function, relates in particular to a kind of DNMT1 and knocks out Mus and can improve the New function of Foxb2 protein expression level.
Background technology
Dnmt rna 1, i.e. DNMT1, be the key enzyme of a kind of catalytic dna cytosine methylation.DNA methylation is the important modification means of life entity impression external world's input signal, adjusted and controlled gene expression, is one of the main category of epigenetics.DNMT1 has important effect in the duplication reparation maintaining DNA methylation feature and DNA.DNMT1 is as the modulin of a kind of gene expression, and its difference that target gene is selected under different physiological conditions eventually affects cell and the change of tissue allomeric function.
Transgenic animal refer to by the animal varieties that engineered means make some gene delection or increment express and obtain, wherein transgenic mice, also known as genetic engineering Mus, in the research of the subjects such as modern neuro biology, pathology, toxicology, play irreplaceable effect.The effect of target protein and the regulating and controlling effect of epigenetics are all had very big value by the interaction between research albumen, regulatory factor by genetic engineering Mus.
Fox albumen is a class transcription factor, mainly plays important regulating and controlling effect in the gene expression relevant to Growth of Cells, breeding, differentiation and life etc..Considerable Fox albumen works in fetal development, and Fox albumen can combine with the chromatin after tightening in cell differentiation procedure.Foxb2 is one of member of Fox albumen, and it is using typical " jaw " structure as DNA binding domain, and it, as the downstream molecules of the signal paths such as hedgehog, has in the growth course of central nervous system and play the part of certain role.
Summary of the invention
Genetic engineering Mus used herein, has namely knocked out the C57BL/6J mice of DNMT1 gene, purchased from only Shang Lide bio tech ltd, Beijing.For the C57BL/6J mice that compares purchased from Medical University Of Anhui.
Having knocked out the C57BL/6J mice of DNMT1 gene, namely described " genetic engineering Mus ", the expression of its corticocerebral Foxb2 albumen brings up to normal compare Mus 2.7 times.
The process of protein extraction and detection is as follows:
By each for the C57BL/6J mice of the genetic engineering Mus and comparison that are cultured to 1 month 6 brain cortical tissues put to death and take out its left brain respectively.Carry out homogenate with refiner after above-mentioned cortical tissue being cut into small pieces with eye scissors, fully grind, by tissue homogenate.Being proceeded to by homogenate in the centrifuge tube of 1.5ml, 12000rpm is 4oCentrifugal 15min under C.Liquid in centrifuge tube divides three layers, colourless liquid phase in the middle of extracting, and proceeds in new centrifuge tube.
The BCA method albumen of extraction first passing through this area conventional measures its protein concentration.Polyacrylamide gel electrophoresis is carried out subsequently with after the isopyknic 2 SDS sample-loading buffer mixing taken advantage of.The condition of electrophoresis is set as voltage 120V, electrophoresis time 1.5h.Carrying out transferring film with nitrocellulose filter subsequently, transferring film condition is 60V transferase 12 h.To be soaked in the solution containing Foxb2 antibody after Membrane cleaning 4oC overnight incubation.With after after repeatedly cleaning, use that commonly used in the art two are anti-hatches, finally carry out chemical development, complete protein blot experiment.The density value of the development band obtained measures and mutual comparison uses ImageJ software to complete.
The expression of the Foxb2 albumen that experimental group and matched group obtain relatively as shown in Figure 1.
From figure 1 it appears that the expression of the Foxb2 albumen of genetic engineering Mus is 2.7 times of comparison Mus.
The application confirms that the expression of Foxb2 is had significant regulating and controlling effect by DNMT1 in central nervous system of mice, the disappearance indicating DNMT1 result in the abnormal expression including the elements such as transcription factor, it was demonstrated that DNMT1 plays indispensable regulating and controlling effect in nervous system development.
Accompanying drawing explanation
The expression of Fig. 1 transgenic mouse and the comparison corticocerebral Foxb2 albumen of Mus
Control refers to comparison Mus；DNMT-KO refers to genetic engineering Mus described herein.
Detailed description of the invention
Embodiment 1
The process of protein extraction and detection is as follows:
By each for the C57BL/6J mice of the genetic engineering Mus and comparison that are cultured to 1 month 6 brain cortical tissues put to death and take out its left brain respectively.Carry out homogenate with refiner after above-mentioned cortical tissue being cut into small pieces with eye scissors, fully grind, by tissue homogenate.Being proceeded to by homogenate in the centrifuge tube of 1.5ml, 12000rpm is 4oCentrifugal 15min under C.Liquid in centrifuge tube divides three layers, colourless liquid phase in the middle of extracting, and proceeds in new centrifuge tube.
The BCA method albumen of extraction first passing through this area conventional measures its protein concentration.Polyacrylamide gel electrophoresis is carried out subsequently with after the isopyknic 2 SDS sample-loading buffer mixing taken advantage of.The condition of electrophoresis is set as voltage 120V, electrophoresis time 1.5h.Carrying out transferring film with nitrocellulose filter subsequently, transferring film condition is 60V transferase 12 h.To be soaked in the solution containing Foxb2 antibody after Membrane cleaning 4oC overnight incubation.With after use two anti-to hatch after repeatedly cleaning, finally carry out chemical development, complete protein blot experiment.The density value of the development band obtained measures and mutual comparison uses ImageJ software to complete.
Claims (4)
1. the New function of a genetic engineering Mus, it is characterised in that it can cause the change of transcription factor expression level.
2. the New function of a genetic engineering Mus, it is characterised in that the expression of its Foxb2 albumen is significantly improved than normal mouse.
3. the New function of genetic engineering Mus as claimed in claim 2, it is characterised in that this genetic engineering Mus is the C57BL/6J mice after DNMT1 gene knockout.
4. the New function of genetic engineering Mus as claimed in claim 3, it is characterised in that the expression of its Foxb2 albumen is 2.7 times of comparison Mus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610162649.2A CN105766795A (en) | 2016-03-22 | 2016-03-22 | New function of genetically engineered mouse for improving Foxb2 expression level |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610162649.2A CN105766795A (en) | 2016-03-22 | 2016-03-22 | New function of genetically engineered mouse for improving Foxb2 expression level |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105766795A true CN105766795A (en) | 2016-07-20 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610162649.2A Pending CN105766795A (en) | 2016-03-22 | 2016-03-22 | New function of genetically engineered mouse for improving Foxb2 expression level |
Country Status (1)
| Country | Link |
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| CN (1) | CN105766795A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016027794A1 (en) * | 2014-08-18 | 2016-02-25 | 和光純薬工業株式会社 | Cancer marker and cancer determination method |
-
2016
- 2016-03-22 CN CN201610162649.2A patent/CN105766795A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016027794A1 (en) * | 2014-08-18 | 2016-02-25 | 和光純薬工業株式会社 | Cancer marker and cancer determination method |
Non-Patent Citations (2)
| Title |
|---|
| 章晓燕等: "FOXC2基因与肥胖", 《国外医学内分泌分册》 * |
| 陶欣: "DNA甲基化转移酶1（DNMT1）在胰腺癌中表达及意义", 《中国优秀硕士学位论文全文数据库》 * |
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Application publication date: 20160720 |
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