CN106520975A - Method for judging reliability of MicroRNA gene knock-out animal serving as experimental animal model - Google Patents

Method for judging reliability of MicroRNA gene knock-out animal serving as experimental animal model Download PDF

Info

Publication number
CN106520975A
CN106520975A CN201611061183.3A CN201611061183A CN106520975A CN 106520975 A CN106520975 A CN 106520975A CN 201611061183 A CN201611061183 A CN 201611061183A CN 106520975 A CN106520975 A CN 106520975A
Authority
CN
China
Prior art keywords
mir
microrna
gene
minutes
gene knock
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201611061183.3A
Other languages
Chinese (zh)
Inventor
王万晨
郁多男
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yangzhou University
Original Assignee
Yangzhou University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yangzhou University filed Critical Yangzhou University
Priority to CN201611061183.3A priority Critical patent/CN106520975A/en
Publication of CN106520975A publication Critical patent/CN106520975A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knockout animals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/25—Animals on a special diet
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases

Abstract

The invention discloses a method for judging the reliability of a MicroRNA gene knock-out animal serving as an experimental animal model, and belongs to the field of experimental model evaluation. MicroRNA-451 containing food is fed to a MicroRNA gene knock-out type mouse, and then the MicroRNA is detected in the body of the mouse. After the mouse is fed, the oxidation resistance of erythrocytes of the mouse is enhanced, which expresses that food-borne MicroRNA can enter the body of the mouse through a digestive tract and play a function, so that the interference of the food-borne MicroRNA-451 of an mm-451 gene knock-out type mouse cannot be excluded, and the reliability is not high. The method for judging the reliability of the MicroRNA gene knock-out animal serving as the experimental animal model provided by the invention is widely applied to research institutions and enterprises which carry out related experiments by using the MicroRNA gene knock-out animal, and has a great scientific value and great economic benefits.

Description

A kind of reliability of judgement MircroRNA Gene Knock-Out Animal Models as experimental animal model The method of property
Technical field
The present invention relates to a kind of side of judgement MircroRNA Gene Knock-Out Animal Models as the reliability of experimental animal model Method, belongs to experimental model evaluation areas.
Background technology
Since nearly more than ten year, microRNA (miRNA) is always the study hotspot of biology field, has now been found that MiRNAs is that a class forms loop-stem structure precursor by non-protein encoding gene transcript, and the length formed Jing after enzyme shears maturation is The small RNA molecular of 18～25 nucleotide.MiRNAs can participate in the Eukaryotic gene expression of regulation and control in post-transcriptional level, its By incomplete base complementrity, specific target gene is acted on, form powerful regulated and control network, cause target gene stability to drop Low or expression is reduced, and plays very important work during development, propagation, differentiation and cell death in various cells etc. With.
Academic circles at present is absorbed for can the MicroRNA of food source enter trencherman and still suffers from dispute.Existing experiment Method predominantly contains the food or synthesis MicroRNA of certain specific MicroRNA to mouse feeding, afterwards by mouse blood MicroRNA reverse transcriptions be DNA, detected using polymerase chain reaction.Existing method is primarily present following deficiency：A, mice If there is the MicroRNA with the MicroRNA sequence similarities of food source in vivo, testing result can be interfered, be affected As a result accuracy.When b, use synthesis MicroRNA, experimental cost is high, the cycle is long.
Although academia has the research carried out with MicroRNA Gene Knock-Out Animal Models as model in a large number.But, for such The reliability of model, is particularly knocked whether gene still appears in this problem in knock out mice, still lacks letter Single effectively evaluation measures.
The content of the invention
The technical problem to be solved is to provide one kind and judges that MircroRNA Gene Knock-Out Animal Models are dynamic as experiment The method of the reliability of thing model, by the MicroRNA Gene Knock-Out Animal Model specific foods that feeding is to be verified, after certain hour Animal blood sample after feeding is taken out, whether detection wherein has the MicroRNA being knocked.
The present invention provides a kind of method of accurate, quick, the economic MicroRNA absorbabilities for judging food source, feeds After food of the food miR-144/451-/- gene knockout type mice rich in MicroRNA-451, in miR-144/451-/- clpp gene Except MicroRNA-451 (blood of the food of feeding for wild-type mice) is detected in type mice body, solve in prior art The problem of presence, it is significant to the laboratory of all use MicroRNA Gene Knock-Out Animal Models, with stronger market reality Shi Xing.
The concrete technical scheme of the present invention is as follows：One kind judges MircroRNA Gene Knock-Out Animal Models as laboratory animal mould The method of the reliability of type, it is characterised in that comprise the following steps:
1) get out related experiment reagent；
2) male mice of wild-type mice and miR-144/451 gene knockouts is each two, obtains substantial amounts of by breeding Wild-type mice and miR-144/451 clpp gene deratization；
3) mice, humidity are raised according to SPF (Specific Pathogen Free no-special pathogens) level feeding standard In 40%-70%, temperature control changes two to three in one week at 20-25 DEG C with the feedstuff of sterilization treatment, bedding and padding and drinking-water for control It is secondary；
4) male Mus and 1-5 miR-144/451 gene knockout that mode is 1 miR-144/451 gene knockout are mated Raettin copulation is bred, it is found that raettin has pregnancy performance to divide cage to give birth to immediately, and record gives birth to the mice father and mother system in time Source；
5) step 4) in mouse DNA extract:The mouse tail end flap of 3 week old is taken, length is 0.5cm, is put into In 1.5ml EP pipes, often pipe plus l00 μ l lysates, stand 20 minutes by 95 DEG C, add l00 μ l neutralizers to mix after cooling, take with On the supernatant containing Mus DNA carry out murine genes type identification for PCR (polymerase chain reaction)；
6) PCR of mouse DNA：Following reactants constitute the PCR reaction systems of 20 μ l：2 μ l of supernatant containing Mus DNA, draw 1 μ l of thing, 10 μ l of archaeal dna polymerase mixture, 7 μ l of distilled water；Above-mentioned material is placed in PCR instrument and imposes a condition to circulate and expands after mixing centrifugation Increase：Denaturation, 95 DEG C, 5 minutes；Degeneration, 95 DEG C, 40 seconds；Annealing, 60.5 DEG C, 40 seconds；Extend, 72 DEG C, 1min；Circulation, 72 DEG C, 5 minutes；
7) row agarose gel electrophoresis are entered：Take in the TAE electrophoretic buffers that agarose powder 0.75g adds 50ml, microwave Boiling is heated in stove, the agar thing room temperature of dissolving clarification is cooled to into 60 DEG C and adds 3 μ l of Goldview5 μ l or EB to mix, delayed Slow being poured into put in the offset plate of comb, and room temperature places 30min, gently extracts comb after gel solidifies completely；To prepare 1.5% agarose gel be put into electrophoresis tank, add 1 × TAE solution to being higher by gel surface；Take 10 μ l of PCR reacting final products It is loaded onto in glue hole, voltage 100V electrophoresis, the time is 30min；
8) gel imaging：After electrophoresis terminates, taking-up gel is placed under gel image analyser takes pictures, mice electrophoresis genotype Fragment is：MiR-144/451 wild-type mices are 290bp, homozygote 190bpheterozygous (- /+) 250bp and 190bp；
9) feeding：Taken from the little angular vein of miR-144/451 wild-type mices using the capillary glass tube with anticoagulant The blood of wild-type mice is fed to miR-144/451 gene knockout type mices using 12# irrigation stomach devices by 200 microlitres of blood；
10) extract sample：Behind feeding 3 hours, 6 hours, 12 hours, 24 hours, respectively using the glass with anticoagulant Glass capillary tube takes 200 microlitres of blood from the little angular vein of miR-144/451 gene knockout type mices；
11) extract the RNA in sample：Per 0.1mL steps 10) whole blood that obtains adds lysate Trizolreagent 1ml, is mixed energetically, stands 5 to 10 minutes；200 μ l chloroforms are added, 15s is acutely rocked, 5 minutes are stood；4 DEG C, 12000g centrifugations 15min；Supernatant is proceeded to into new EP pipes, 500 μ l isopropanols is added, is overturned precipitation at room temperature 10min after mixing；4 DEG C, 12000g from Heart 20min；Supernatant is abandoned, 1ml75% ethanol is added；4 DEG C, 12000g centrifugation 5min；Supernatant is abandoned, is stored at room temperature to dry and is added 20 μ lDEPC water dissolutioies；55 DEG C of water-bath 5min；
12) reverse transcription RNA:Configuration reactant liquor system：Step 11) in obtain total serum IgE 1ng, RTase Mix1 μ l, 5 μ l of 5xReaction Buffer, using ddH2Reaction system is complemented to 25 μ l by O；37 DEG C are heated up to after mixing, are then incubated 60 minutes, then 85 DEG C are heated up to, it is incubated 10 minutes；
13) PCR of whole blood DNA：Following reactants constitute the PCR reaction systems of 20 μ l：Step 12) whole blood DNA that obtains 2 μ l, 2 μ l of forward primer, 2 μ l of downstream primer, 10 μ l of archaeal dna polymerase mixture, 7 μ l of distilled water；PCR instrument is placed in after mixing centrifugation Impose a condition cyclic amplification：Denaturation, 95 DEG C, 5 minutes；Degeneration, 95 DEG C, 40 seconds；Annealing, 60.5 DEG C, 40 seconds；Extend, 72 DEG C, 1min；Circulation, 72 DEG C, 5 minutes；
14) use U6 as internal reference, judge the expression of miR-451.
Step 7) in, the TAE electrophoretic buffers of 50ml are dense by Tris-base, 100ml of the glacial acetic acid of 57.1ml, 242g Spend the EDTA for 0.5mol/L to be formulated, and 1L is settled to by distilled water.
Step 7) in, 1 × TAE electrophoretic buffers add distilled water to be settled to 500ml by the 50xTAE electrophoretic buffers of 10ml.
Step 7) in, the EB concentration is 10mg/ml, mixes lucifuge after dissolving by the deionized water of EB, 100ml of 1g and protects Deposit, room temperature is placed and made.
Step 5) in, 0.5M EDTA of the lysate by 40 μ l, the distilled water system of 10N NaOH, 100ml of 25 μ l Into, and PH is adjusted to 8.0.
Step 5) in, the neutralizer adds distilled water to be settled to 100ml by the 1M Tris-HCl of 4ml.
The present invention is by providing a kind of side of accurate, quick, the economic MicroRNA absorbabilities for judging food source Whether method, the MicroRNA Gene Knock-Out Animal Models that can be used with auxiliary judgment laboratory are reliable.Main implementation is as follows：
1. the mice of the specific MicroRNA of gene knockout is cultivated；
2., by consulting literatures, the animal vegetable tissue of this kind of MicroRNA rich content is found；
3. above-mentioned animal vegetable tissue is made into food and be fed to specific MicroRNA gene knockouts type mice；
4., three hours after feeding, six hours, 12 hours, twenty four hours taking-up mice blood, detect therein MicroRNA contents.
Compared to prior art, beneficial effects of the present invention are：
After by feeding MicroRNA Gene Knock-Out Animal Model specific foods to be verified, after taking out feeding after certain hour Animal blood sample, detects the MicroRNA for being knocked coded by said gene wherein.It is concluded that：It is knocked MicroRNA can be entered in MicroRNA Gene Knock-Out Animal Model bodies by food, and MicroRNA Gene Knock-Out Animal Models are used as experiment The reliability of animal model is not high.
Food containing MicroRNA-451 is fed to MicroRNA-451 gene knockout type mices by the present invention, is existed then This kind of MicroRNA is detected in mice body.After feeding, the oxidation resistance of mouse red blood cell strengthens, and illustrates food-borne MicroRNA can be entered in mice body by digestive tract, and function, therefore mm-451 gene knockout type mices can not be arranged Except the interference of food-borne MicroRNA-451, reliability is not high.The present invention provides one kind and judges MircroRNA Gene Knock-Out Animal Models As the method for the reliability of experimental animal model, the research of related experiment will be carried out using MicroRNA Gene Knock-Out Animal Models Mechanism and enterprise extensively apply, with great scientific value and economic benefit.
Description of the drawings
Fig. 1 is after feeding is rich in MicroRNA-451 blood, in miR-144/451-/- gene knockout type mice body MicroRNA-451 changes of contents situations；
Fig. 2 is genotype mice identification gel electrophoresis figure.
Specific embodiment
A kind of experimental technique for judging miR-144/451-/- gene knockout type according to this method, comprises the following steps：
S1：Get out related experiment reagent
(1) experiment reagent that the present embodiment is related to is as shown in the table：
(2) the main Self-made reagent that the present embodiment is related to is as shown in the table：
(3) PCR primer being applied in the present embodiment：
(3.1) real-time fluorescence quantitative PCR primer：
5 '-AAACCGTTACCATTACTGAGTT-3 ' of miR-451 forward primer
Downstream primer is logical in GeneCopoeia companies All-in-One miRNA qRT-PCR Detection Kit Use primer.
(3.2) genotype identification primer：
MiR451 forward primer：5’-TTCTGCCTGTAACTCTGGATCCCTAAGAGA
MiR451 downstream primers -1：5’-GGGTACCCAGACTAGTACATCATCTATA
MiR451 downstream primers -2：5’-ATCCCCTCGAGGGACCTAATAACTTC
S2：Yangzhou University's non-coding RNA center by attached The Children's Hospital of Philadelphia of Univ Pennsylvania USA introduce β- Globin- /+each two with miR-144/451-/- gene knockout male mice, Yangzhou University's Correlation Centre C57/BL6 females are little Some of Mus, have bred at present and have obtained substantial amounts of gene knockout β-thalassemia Mus, the deratization of miR-144/451 clpp genes and The lean mouse model in β-ground of miR-144/451 gene knockouts.
S3：Mice is raised according to SPF (Specific Pathogen Free no-special pathogens) level feeding standard.Humidity In 40%-70%, temperature control changes two to three in one week at 20-25 DEG C with the feedstuff of sterilization treatment, bedding and padding and drinking-water for control It is secondary.
S4：The mode of mating is that the male Mus of 1 gene knockout and the raettin copulation of 1-5 gene knockout are bred, and is found Raettin has pregnancy performance such as cloudy bolt or abdominal part to resemble a pear in shape cage fertility the record mice father's maternity in time of divide immediately.
S5：Mouse DNA is extracted：The mouse tail end flap of 3 week old is taken, size about 0.5cm is put into 1.5ml EP pipes In, often pipe adds l00 μ l lysates, 95 DEG C of standing 20min.Add l00 μ l neutralizers to mix after cooling, take the DNA containing Mus of the above Supernatant carry out murine genes type identification for PCR.
S6：The polymerase chain reaction (PCR) of mouse DNA：Primer is provided by Shanghai Sheng Gong biological engineering company limited. PCR reaction systems：Following reactants constitute the reaction system of 20 μ l.2 μ l of rat-tail DNA, primer (20 μm of ol) μ 1, archaeal dna polymerase 10 μ l of mixture (Taq archaeal dna polymerases, dNTPs, MgCl2 and reaction buffer), 7 μ l of distilled water.PCR is placed in after mixing centrifugation Instrument imposes a condition cyclic amplification：Denaturation, 95 DEG C, 5 minutes；Degeneration, 95 DEG C, 40 seconds；Annealing, 60.5 DEG C, 40 seconds；Extend, 72 DEG C, 1 minute；Circulation；72 DEG C, 5 minutes.
S7：Enter row agarose gel electrophoresis：Take in the TAE electrophoretic buffers that agarose powder 0.75g adds 50ml, microwave Be heated in stove boiling, by dissolving clarification agar thing room temperature be cooled to 60 DEG C add Goldview 5 μ l or EB 3 μ l mix, Slowly pour into and put in the offset plate of comb, room temperature is placed 30min and gently extracts comb after gel solidifies completely.To prepare 1.5% agarose gel be put into electrophoresis tank, add 1 × TAE solution to being higher by gel surface.Take 10 μ l of PCR reacting final products It is loaded onto in glue hole.Voltage 100V electrophoresis, time are 30 minutes.
S8：Gel imaging：After electrophoresis terminates, taking-up gel is placed under gel image analyser takes pictures, mice electrophoresis gene Matrix section is：β-thalassemia mice identification is that a size is wild type for the band of 567bp (base pair, base) Normal mice, a size are homozygote β-thalassemia mice for the band of 1000bp；Occur more than two band for β- Thalassemia heterozygote.MiR-144/451 wild-type mices are 290bp；Homozygote 190bp；Heterozygote (- /+) 250bp and 190bp。
S9：Feeding：Taken from the little angular vein of miR-144/451 wild-type mices using the capillary glass tube with anticoagulant The blood of wild-type mice is fed to miR-144/451 gene knockout type mices using 12# irrigation stomach devices by 200 microlitres of blood；
S10：Extract sample：Behind feeding 3 hours, 6 hours, 12 hours, 24 hours, respectively using the glass with anticoagulant Glass capillary tube takes 200 microlitres of blood from the little angular vein of miR-144/451 gene knockout type mices；
S11：Extract the RNA in sample：Per 0.1mL steps 10) whole blood that obtains adds lysate Trizolreagent 1ml, is mixed energetically, stands 5 to 10 minutes；200 μ l chloroforms are added, 15s is acutely rocked, 5 minutes are stood；4 DEG C, 12000g centrifugations 15min；Supernatant is proceeded to into new EP pipes, 500 μ l isopropanols is added, is overturned precipitation at room temperature 10min after mixing；4 DEG C, 12000g from Heart 20min；Supernatant is abandoned, 1ml75% ethanol is added；4 DEG C, 12000g centrifugation 5min；Supernatant is abandoned, is stored at room temperature to dry and is added 20 μ lDEPC water dissolutioies；55 DEG C of water-bath 5min；
S12：Reverse transcription RNA:Configuration reactant liquor system：Step 11) in obtain 1 μ l of total serum IgE 1ng, RTase Mix, 5 μ l of 5xReaction Buffer, using ddH2Reaction system is complemented to 25 μ l by O；37 DEG C are heated up to after mixing, are then incubated 60 minutes, then 85 DEG C are heated up to, it is incubated 10 minutes；
S13:The PCR of whole blood DNA：Following reactants constitute the PCR reaction systems of 20 μ l：Step 12) whole blood DNA that obtains 2 μ l, 2 μ l of forward primer, 2 μ l of downstream primer, 10 μ l of archaeal dna polymerase mixture, 7 μ l of distilled water；PCR instrument is placed in after mixing centrifugation Impose a condition cyclic amplification：Denaturation, 95 DEG C, 5 minutes；Degeneration, 95 DEG C, 40 seconds；Annealing, 60.5 DEG C, 40 seconds；Extend, 72 DEG C, 1min；Circulation, 72 DEG C, 5 minutes；
S14:Using U6 as internal reference, the expression of miR-451 is judged.

Claims (6)

1. method of a kind of judgement MircroRNA Gene Knock-Out Animal Models as the reliability of experimental animal model, it is characterised in that Comprise the following steps:
1）Get out related experiment reagent；
2）The male mice of wild-type mice and miR-144/451 gene knockouts is each two, obtains substantial amounts of wild by breeding Type mice and miR-144/451 clpp gene deratization；
3）Mice is raised according to SPF levels feeding standard, humid control in 40%-70%, temperature control at 20-25 DEG C, with sterilizing The feedstuff of process, bedding and padding and drinking-water are changed two to three times for one week；
4）The mode of mating is the raettin of the male Mus of 1 miR-144/451 gene knockout and 1-5 miR-144/451 gene knockout Copulation is bred, it is found that raettin has pregnancy performance to divide cage to give birth to immediately, and record gives birth to the mice father's maternity in time；
5）Step 4）In mouse DNA extract:The mouse tail end flap of 3 week old is taken, length is 0.5cm, is put into 1.5ml In EP pipes, often pipe plus l00 μ l lysates, stand 20 minutes by 95 DEG C, add l00 μ l neutralizers to mix, take containing for the above after cooling The supernatant of Mus DNA carries out murine genes type identification for PCR；
6）The PCR of mouse DNA：Following reactants constitute the PCR reaction systems of 20 μ l：2 μ l of supernatant containing Mus DNA, 1 μ of primer L, 10 μ l of archaeal dna polymerase mixture, 7 μ l of distilled water；Above-mentioned material is placed in PCR instrument after mixing centrifugation and imposes a condition cyclic amplification： Denaturation, 95 DEG C, 5 minutes；Degeneration, 95 DEG C, 40 seconds；Annealing, 60.5 DEG C, 40 seconds；Extend, 72 DEG C, 1min；Circulation, 72 DEG C, 5 Minute；
7）Enter row agarose gel electrophoresis：Take in the TAE electrophoretic buffers that agarose powder 0.75g adds 50ml, in microwave oven Be heated to boiling, by dissolving clarification agar thing room temperature be cooled to 60 DEG C add Goldview 5 μ l or EB 3 μ l mix, slowly Pour into, room temperature places 30min, gently extracts comb after gel solidifies completely；By what is prepared 1.5% agarose gel is put into electrophoresis tank, adds 1 × TAE solution to being higher by gel surface；Take 10 μ l of PCR reacting final products sample-addings To in glue hole, voltage 100V electrophoresis, the time is 30min；
8）Gel imaging：After electrophoresis terminates, taking-up gel is placed under gel image analyser takes pictures, mice electrophoresis genotype fragment For：MiR-144/451 wild-type mices are 290bp, homozygote 190bpheterozygous (- /+) 250bp and 190bp；
9）Feeding：Blood 200 is taken using the capillary glass tube with anticoagulant from the little angular vein of miR-144/451 wild-type mices Microlitre, the blood of wild-type mice is fed to into miR-144/451 gene knockout type mices using 12# irrigation stomach devices；
10）Extract sample：Behind feeding 3 hours, 6 hours, 12 hours, 24 hours, respectively using the glass fiber with anticoagulant Tubule takes 200 microlitres of blood from the little angular vein of miR-144/451 gene knockout type mices；
11）Extract the RNA in sample：Per 0.1mL steps 10）The whole blood for obtaining adds 1 ml of lysate Trizolreagent, Mix energetically, stand 5 to 10 minutes；200 μ l chloroforms are added, 15s is acutely rocked, 5 minutes are stood；4 DEG C, 12000g centrifugations 15min；Supernatant is proceeded to into new EP pipes, 500 μ l isopropanols is added, is overturned precipitation at room temperature 10min after mixing；4 DEG C, 12000g Centrifugation 20min；Supernatant is abandoned, 1ml75% ethanol is added；4 DEG C, 12000g centrifugation 5min；Supernatant is abandoned, is stored at room temperature to dry addition 20 μ lDEPC water dissolutioies；55 DEG C of water-bath 5min；
12）Reverse transcription RNA:Configuration reactant liquor system：Step 11）In obtain 1 ng, RTase Mix of total serum IgE, 1 μ l, 5 μ l of 5xReaction Buffer, using ddH2Reaction system is complemented to 25 μ l by O；37 DEG C are heated up to after mixing, are then incubated 60 minutes, then 85 DEG C are heated up to, it is incubated 10 minutes；
13）The PCR of whole blood DNA：Following reactants constitute the PCR reaction systems of 20 μ l：Step 12）The 2 μ l of whole blood DNA that obtain, 2 μ l of forward primer, 2 μ l of downstream primer, 10 μ l of archaeal dna polymerase mixture, 7 μ l of distilled water；PCR instrument is placed in after mixing centrifugation to set Fixed condition cyclic amplification：Denaturation, 95 DEG C, 5 minutes；Degeneration, 95 DEG C, 40 seconds；Annealing, 60.5 DEG C, 40 seconds；Extend, 72 DEG C, 1min；Circulation, 72 DEG C, 5 minutes；
14）Using U6 as internal reference, the expression of miR-451 is judged.
2. judgement MircroRNA Gene Knock-Out Animal Models according to claim 1 are used as the reliability of experimental animal model Method, is characterized in that, step 7）In, the TAE electrophoretic buffers of 50ml by the glacial acetic acid of 57.1ml, the Tris-base of 242g, 100ml concentration is formulated for the EDTA of 0.5mol/L, and is settled to 1L by distilled water.
3. judgement MircroRNA Gene Knock-Out Animal Models according to claim 1 are used as the reliability of experimental animal model Method, is characterized in that, step 7）In, 1 × TAE electrophoretic buffers add distilled water to be settled to by the 50xTAE electrophoretic buffers of 10ml 500ml。
4. judgement MircroRNA Gene Knock-Out Animal Models according to claim 1 are used as the reliability of experimental animal model Method, is characterized in that, step 7）In, the EB concentration is 10mg/ml, is mixed after dissolving by the deionized water of EB, 100ml of 1g Keep in dark place, room temperature is placed and made.
5. judgement MircroRNA Gene Knock-Out Animal Models according to claim 1 are used as the reliability of experimental animal model Method, is characterized in that, step 5）In, 0.5M EDTA of the lysate by 40 μ l, the distillation of 10N NaOH, 100ml of 25 μ l Water is made, and adjusts PH to 8.0.
6. judgement MircroRNA Gene Knock-Out Animal Models according to claim 1 are used as the reliability of experimental animal model Method, is characterized in that, step 5）In, the neutralizer adds distilled water to be settled to 100ml by the 1M Tris-HCl of 4ml.
CN201611061183.3A 2016-11-28 2016-11-28 Method for judging reliability of MicroRNA gene knock-out animal serving as experimental animal model Pending CN106520975A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201611061183.3A CN106520975A (en) 2016-11-28 2016-11-28 Method for judging reliability of MicroRNA gene knock-out animal serving as experimental animal model

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201611061183.3A CN106520975A (en) 2016-11-28 2016-11-28 Method for judging reliability of MicroRNA gene knock-out animal serving as experimental animal model

Publications (1)

Publication Number Publication Date
CN106520975A true CN106520975A (en) 2017-03-22

Family

ID=58356909

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201611061183.3A Pending CN106520975A (en) 2016-11-28 2016-11-28 Method for judging reliability of MicroRNA gene knock-out animal serving as experimental animal model

Country Status (1)

Country Link
CN (1) CN106520975A (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101886115A (en) * 2009-05-11 2010-11-17 中国医学科学院基础医学研究所 Application of three microRNAs in diagnosis and treatment of human acute myeloid leukemia
CN103642836A (en) * 2013-11-26 2014-03-19 苏州同善生物科技有限公司 Method for establishing fragile X-syndrome non-human primate model on basis of CRISPR gene knockout technology
CN103911436A (en) * 2014-03-13 2014-07-09 南京医科大学 Serum/plasma miRNA marker for early diagnosis of noncardiac gastric carcinoma and applications thereof
CN105854017A (en) * 2016-03-01 2016-08-17 扬州大学 Reagent for treating beta-thalassemia and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101886115A (en) * 2009-05-11 2010-11-17 中国医学科学院基础医学研究所 Application of three microRNAs in diagnosis and treatment of human acute myeloid leukemia
CN103642836A (en) * 2013-11-26 2014-03-19 苏州同善生物科技有限公司 Method for establishing fragile X-syndrome non-human primate model on basis of CRISPR gene knockout technology
CN103911436A (en) * 2014-03-13 2014-07-09 南京医科大学 Serum/plasma miRNA marker for early diagnosis of noncardiac gastric carcinoma and applications thereof
CN105854017A (en) * 2016-03-01 2016-08-17 扬州大学 Reagent for treating beta-thalassemia and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GAOFENG LIANG等: "Assessing the survival of exogenous plant microRNA in mice", 《FOOD SCIENCE & NUTRITION》 *

Similar Documents

Publication Publication Date Title
Kobor et al. Focus on: epigenetics and fetal alcohol spectrum disorders
Sevane et al. Genome‐wide differential DNA methylation in tropically adapted Creole cattle and their Iberian ancestors
CN108893544B (en) SNP molecular marker related to litter size of pig menstruation, identification and application thereof
CN108546766B (en) SNP molecular marker related to pig litter traits, identification and combined application thereof
CN106939333B (en) Application of the miRNA marker in the Cervical intraepitheliaI neoplasia reagent for preparing screening folic acid deficiency group
Brevik et al. Paternal benzo [a] pyrene exposure modulates microRNA expression patterns in the developing mouse embryo
CN104109714A (en) Primer for detecting folic acid metabolism related gene SNP, fluorescent probe and application
He et al. Identification and characterization of microRNAs in the gonad of Trachinotus ovatus using Solexa sequencing
CN105854017A (en) Reagent for treating beta-thalassemia and application thereof
Finot et al. Reference gene selection for quantitative real-time PCR normalization: application in the caprine mammary gland
Tang et al. Identification of loci affecting teat number by genome-wide association studies on three pig populations
CN114657264B (en) Clarias fuscus sex-specific molecular marker primer and application thereof
CN103789406B (en) A kind of PCR-RFLP method detecting cattle Pax3 gene mononucleotide polymorphism and application
CN113699152A (en) Construction method and application of SLC35E2B gene knockout mouse animal model
CN103898231A (en) SNP (Single Nucleotide Polymorphism) molecular marker related to pork pH characteristics and application thereof
Wang et al. Expression profile analysis of sheep ovary after superovulation and estrus synchronisation treatment
CN112921101A (en) Molecular marker related to sheep remaining feed intake and application thereof
CN106801100A (en) A kind of method and its application based on ACACB identified for genes cow producing milk proterties
Li et al. Comprehensive analysis of circRNAs expression profiles in different periods of MDBK cells infected with bovine viral diarrhea virus
CN106520975A (en) Method for judging reliability of MicroRNA gene knock-out animal serving as experimental animal model
Kaushik et al. Molecular characterization and expression profiling of ENOX2 gene in response to heat stress in goats
CN102344959A (en) Nucleic acid detection
CN104232643A (en) RNAi (ribonucleic acid interference) interference segment, interference vector, and preparation method and application thereof
Xie et al. Impact of FecB mutation on ovarian DNA methylome in small-tail Han sheep
Magalhães et al. Placental hydroxymethylation vsmethylation at the imprinting control region 2 on chromosome 11p15. 5

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20170322