CN106755094A - Mouse model and build and application that IRTKS genes liver specificity is rejected - Google Patents
Mouse model and build and application that IRTKS genes liver specificity is rejected Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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Abstract
The mouse model and structure rejected the invention discloses a kind of IRTKS genes liver specificity and application；By homologous recombination, the sequence containing exon2 in mouse IRTKS genes is instead of with 2.245 kb frt neo frt loxp Flox loxp sequences, obtain IRTKSfl。x/+Allophenic mice；Using Cre recombinases, the allophenic mice for obtaining is mated with the mouse Alb Cre of liver specific expression Cre, it is final to obtain the mouse model that IRTKS genes liver specificity is rejected.The IRTKS gene knockout mice models that the above method builds can be used for diabetes study；By body weight, fasting blood-glucose, sugar tolerance and pathological analysis, its feature is consistent with the onset diabetes feature of the mankind, so that for the research of pathogenesis of diabetes mellitus and the evaluation of medicine and screening provide good animal model.
Description
Technical field
It is to be related to a kind of IRTKS genes liver more specifically the present invention relates to the animal model in biological technical field
The mouse model that characteristic is rejected, its construction method, and its application.
Background technology
Diabetes are that, by inherent cause, one kind that many factors collective effect such as environmental factor and life-form structure causes is with height
The metabolic disease that blood sugar is characterized.The illness rate of diabetes, disability rate and case fatality rate and the extent of injury to general health,
The 3rd of Chronic Non-Communicable Diseases is occupy, the death rate number caused by diabetes and its complication has also occupy the world
The 5th of the cause of death.Studies have reported that world's diabetes number of patients in 2013 has reached 3.82 hundred million, and expect 2030
Year increases to 5.52 hundred million people.Past, with China's rapid economic development, the illness rate of domestic diabetes increased ten over more than 30 years
Several times, the illness rate of the people's survey data display diabetes of national 14 provinces and cities 300,000 is 0.67% within 1980；Investigation display in 2010,
China's crowd's diabetes prevalence of more than 18 years old is 9.7%.According to the data of International Diabetes Federation, China 2014
Diabetes number of patients is 96,290,000 people, occupies global first place.Therefore the high incidence of diabetes causes the great attention in the whole world.
In order to further investigate the pathogenesis of diabetes, good animal model is very important research tool.In early days,
Researcher is chemical by physics, the paathogenic factor such as biology, the manual-induced experimental animal model with diabetic character, from
Hair property heredity diabetes model due to without artificial intervention factor, can more simulate the real pathogenesis of diabetes mellitus of the mankind,
It is applied to pathogenesis of diabetes mellitus and the convenient scientific research value of complication is larger, and more typical has ob/ob, db/db mouse etc.,
It is respectively fat adjoint serious insulin resistance caused by the mutation due to leptin gene or leptin gene acceptor, but these are dynamic
Thing model is limited in that the gene mutation genetic background that it is based on is not common in human patientses.In recent years, with
Researcher gos deep into pathogenesis of diabetes mellitus understanding, and transgenic animal model technology reaches its maturity, based on disease
Related keyword signal path transgenosis or knockout animal model in the research of diabetes using increasing.The opposing party
Face, because diabetes are the clinical syndromes that one group of various genetic background and environmental factor interact and trigger, although at present
The diabetes animal model of exploitation is a lot, but still has deviation with human diabetes, there is no fitted like a glove with human diabetes so far
Animal model.New research progress based on pathogenesis of diabetes mellitus, exploitation obtains the animal mould of preferably simulation human diabetes
Type is still that we explore diabetes, the effective tool of insulin resistance and required Research approach.
The content of the invention
It is an object of the invention to provide a kind of IRTKS genes liver specificity reject mouse model and build with should
With.It can be that the research of diabetes and the discussion of Molecular pathogenesis provide research model.
The purpose of the present invention is achieved through the following technical solutions：
In a first aspect, a kind of construction method of the mouse model rejected the present invention relates to IRTKS genes liver specificity, leads to
The method for crossing homologous recombination, mouse IRTKS genes are instead of with 2.245-kb frt-neo-frt-loxp-Flox-loxp sequences
In the sequence containing exon2, obtain IRTKSflox/+Allophenic mice；The allophenic mice and liver specificity table that will be obtained
Mouse Alb-Cre up to Cre mates, final to obtain the mouse model that IRTKS genes liver specificity is rejected.
Preferably, the construction method is comprised the following specific steps that：
S1, structure IRTKS-CKO plasmids；
S2, ES cytogene are practiced shooting and screening positive clone；
S3, the positive ES clone's injection Mouse Blastocysts, obtain IRTKSflox/+Allophenic mice；
S4, chimera hero mouse and raettin are bred, obtain the positive F1 generation mouse of both arms；
S5, F1 generation mouse or its offspring are mated with Alb-Cre mouse.
Preferably, in step S1, the NotI digestions of IRTKS-CKO DNAs are isolated and purified, absolute ethyl alcohol precipitation, aseptic
PBS is resuspended, obtains IRTKS-CKO plasmids.
Preferably, in step S2, ES cells are SCR012, and colony screening condition is 300 μ g/ml G418 and 2 μM of GanC
Screening 8 days.
Preferably, step S2 also includes identifying that positive ES is cloned with PCR method；The primer pair sequence such as SEQ of 5 ' PCR identifications
ID NO：1st, shown in 2；The primer pair sequence such as SEQ ID NO of 3 ' PCR identifications：3rd, shown in 4.
Preferably, in step S3, the allophenic mice is chimeric male mice of the rate more than 50%.
Preferably, the raettin provided in step S3 in the mouse and step S4 of blastaea is C57BL systems mouse.
Preferably, step S5 also includes identifying genotype with PCR method；Wild type WT PCR's identifies that primer pair is SEQ
ID NO：7th, 8, saltant type Neo PCR's identifies that primer pair is SEQ ID NO：5、6.
Second aspect, what the IRTKS genes liver specificity obtained the invention further relates to a kind of above-mentioned construction method was rejected
Mouse model.
The third aspect, what the IRTKS genes liver specificity obtained the invention further relates to a kind of above-mentioned construction method was rejected
Application of the mouse model in preparing or screening Remedies for diabetes.
Compared with prior art, the present invention has the advantages that：
1) present invention instead of by homologous recombination with 2.245-kb frt-neo-frt-loxp-Flox-loxp sequences
Sequence containing exon2 in mouse IRTKS genes, obtains IRTKSflox/+Allophenic mice.Enzyme viability is recombinated using Cre,
The allophenic mice for obtaining is mated with the mouse Alb-Cre of liver specific expression Cre, reliable IRTKS genes are constructed
The mouse model that liver specificity is rejected.
2) by the mouse model body weight rejected to IRTKS genes liver specificity, fasting blood-glucose, sugar tolerance, and pathology
Detection and analysis, it is found that increased weight occurs in the mouse that IRTKS genes liver specificity is rejected, and hyperglycaemia, glucose tolerance is reduced,
And become with vacuole sample fat in liver section, this is consistent with the pathogeneticing characteristic of the mankind, so as to be pathogenesis of diabetes mellitus
Research and the evaluation of medicine and screening provide good animal model.
Brief description of the drawings
The detailed description made to non-limiting example with reference to the following drawings by reading, further feature of the invention,
Objects and advantages will become more apparent upon：
Fig. 1 is the target practice plasmid construction schematic diagram of the IRTKS-CKO mouse of the embodiment of the present invention 1；Wherein, A is targeting vector
Construction strategy, B for targeting vector schematic diagram, C be IRTKS-CKO target practice schematic diagrames；
Fig. 2 is the genotype identification result schematic diagram of the IRTKS-CKO mouse of the embodiment of the present invention 1；Wherein, for embedding headed by a
The qualification result of 5 ' arm of fit murine genes type, headed by b for allophenic mice genotype 3 ' arm qualification result；
Fig. 3 is the genotype identification strategy of the IRTKS-LKO mouse of the embodiment of the present invention 2, and wherein P5/P6 primers are expanded
Neo is the insertion single allele in loxp sites, and the WT of P7/P8 primers amplification is the single equipotential base for being not inserted into loxp sites
Cause；
Fig. 4 is the genotype PCR qualification results signal that the IRTKS genes liver specificity of the embodiment of the present invention 2 rejects mouse
Figure；
Fig. 5 is the expression of IRTKS mRNA in the quantitative fluorescent PCR of the embodiment of the present invention 2 identification different tissues；
Fig. 6 is that the genotype Western-blot of the IRTKS genes liver specificity of the embodiment of the present invention 2 rejecting mouse is tested
Card result schematic diagram；
Fig. 7 is the male mice body weight contrast of wild type and IRTKS genes liver specificity knockout in the embodiment of the present invention 3
Schematic diagram；
Fig. 8 is the male mice fasting blood-glucose of wild type and IRTKS genes liver specificity knockout in the embodiment of the present invention 3
Value contrast schematic diagram；
Fig. 9 is that the male mice insulin of wild type and IRTKS genes liver specificity knockout in the embodiment of the present invention 3 is resistance to
Amount curve map；
Figure 10 is the male mice liver group of wild type and IRTKS genes liver specificity knockout in the embodiment of the present invention 3
Knit HE colored graphs；Wherein, a is the HE coloration results of WT mouse liver under normal diet, and b is LKO mouse under normal diet
The HE coloration results of liver.
Specific embodiment
With reference to embodiment, the present invention is described in detail.Following examples will be helpful to those skilled in the art
The present invention is further understood, but the invention is not limited in any way.It should be pointed out that to one of ordinary skill in the art
For, without departing from the inventive concept of the premise, can also make certain adjustments and improvements.These belong to guarantor of the invention
Shield scope.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition, such as Sambrook etc.
People, molecular cloning, laboratory manual (New York：Cold Spring Harbor Laboratory Press, 1989) in institute
The condition stated, or according to the condition proposed by manufacturer.
Embodiment 1, the structure of IRTKS-CKO mouse
1st, the structure of IRTKS-CKO plasmids
Referring to Fig. 1, by 100 μ g IRTKS-ABRLFn-pBR322 targeting vectors DNA NotI (enzyme dosages：It is 120U) linear
Change, digestion system be 200 μ l, 37 DEG C digestion overnight, isometric phenol chloroform, chloroform treatment after, absolute ethyl alcohol precipitation, 100 μ l without
Bacterium PBS is resuspended standby.
2nd, ES cell targetings and screening
Above-mentioned IRTKS-ABRLFn-pBR322 vector linearizations plasmid is used for ES cell targetings.
ES cells：SCR012
Linearisation IRTKS-CKO amount of DNA：35μg
Electroporation model：Bio-Rad Gene Pulser(Cat.No.165-2105)
Electroporation conditions：Voltage 240v, electric capacity 500 μ F, actual conduction time 9.6ms, virtual voltage 256v
Colony screening condition：300 μ g/ml G418 and 2 μM of GanC are screened 8 days
Picking resistance clone and totally 96 parts of DNA sample of offer
3rd, the identification of positive ES clones
Two pairs of primers are separately designed, referring to Fig. 1；Wherein, P1/P2 primer pairs identify 5 ' arm, P3/P4 primer pairs identification 3 '
arm。
5 ' PCR are identified：Identification primer P1 IRTKS-5P111 and P2 neo-reverse, purpose fragment is 4188bp；Mirror
Determine result and see Fig. 2.
PCR primer sequence：
P1 IRTKS-5P111：TCTAGACAATGTGCTTGCAGCC(SEQ ID NO：1)
P2 neo reverse：GGCCTACCCGCTTCCATTGCTC(SEQ ID NO：2)
Reaction system (25 μ l)：
PCR reaction conditions：95 DEG C of 4min, 94 DEG C of 45s, 63.6 DEG C of 210s (34cycles), 72 DEG C of 10min.
3 ' PCR are identified：Primer is identified for P3 neo-L and P4 IRTKS-3P211, purpose fragment is 4513bp.Identification knot
Fruit sees Fig. 2.
PCR primer sequence：
P3 neo-Lh：CCGTGCCTTCCTTGACCCTGG(SEQ ID NO：3)
P4 IRTKS-3P211：AACTCAGACGCAGAAACCAGTC(SEQ ID NO：4)
PCR reaction conditions：95℃4min；94 DEG C of 45s, 68 DEG C of 330s (34 cycles), 72 DEG C of 10min.
Reaction system (25 μ l)：
4th, blastaea injection
Microinjection is originated with blastaea：C57BL/6J mouse superfecundation, natural conception is developed to blastocyst stage in vivo.
The embryonic stem cell of positive homologous recombination is injected into the blastaea of C57BL/6J mouse, 96 pieces of embryos of co-injection make 9 and receive
Body.Be born 3 mouse altogether, wherein 3 ♂ are the chimeric hero mouse of ＞ 50%.
5th, allophenic mice is bred
Maturation hero mouse by chimeric rate more than 50% is mated with C57BL/6J raettins, the extracted tail of offspring grey mouse
Genomic DNA enters performing PCR identification (identification strategy is ibid), and both arms positive F1 generation mouse 7 is obtained altogether,
The expression identification of embodiment 2, the genotype identification of IRTKS-LKO mouse and IRTKS
1st, F1 generation mouse is mated breeding with Alb-Cre instrument mouse, murine genes type is identified using PCR, can obtained
IRTKS-LKO mouse.
Two pairs of PCR primers are designed first, referring to Fig. 3.Primer sequence is as follows
Neo(751bp)P5：TAGTTGCCAGCCATCTGTTG(SEQ ID NO：5)
P6：CATCCTGCACTGCTCCTGTA(SEQ ID NO：6)
WT(497bp)P7：CTGGGGTTCCTGAGACTTTG(SEQ ID NO：7)
P8：CATCCTGCACTGCTCCTGTA(SEQ ID NO：8)
And Cre universal primers：(293bp)P9：AACGCTGGTTAGCACCGCAGG(SEQ ID NO：9)
P10：TATCTCGCGCGGCTCCGACA(SEQ ID NO：10)
PCR reaction systems (50 μ l)：The μ l of 0.25 μ l, 10XTaKaRa rTaq buffer solutions of 5U/ul TaKaRa rTaq 5,
The μ l of 2.5mM dNTP mixed liquors 4, DNA profiling 300ng, each 0.3-1 μM of forward and reverse primer, sterile purified water supplies cumulative volume to 50
μl。
PCR reaction conditions：95℃10min；95 DEG C of 10s, 58 DEG C of 30s, 72 DEG C of 30s, totally 30 circulations；72℃5min.
Fig. 4 is PCR qualification result schematic diagrames；Wherein, P7/P8 primer pairs are that identification wild type IRTKS, P5/P6 are identifications
The saltant type in IRTKS-neo insertions loxp sites, shows in figure, and numbering is that 19 murine genes type is IRTKSflox/floxX
Alb-Cre。
2nd, the extraction of mouse tissue RNA, reverse transcription, and quantitative fluorescent PCR
The mouse cervical dislocation crossed through genotype identification is put to death, and is dissected, take each histoorgan (white adipose pad, kidney,
Spleen, pancreas, liver, lung) it is quick-frozen and be transferred to -80 DEG C of Refrigerator stores with liquid nitrogen.Using Trizol reagent
(Invitrogen) tissue RNA is extracted, cDNA is obtained by reverse transcription PCR, finally carry out quantitative fluorescent PCR identification.
Quantitative PCR reaction uses Thermal Cycler Dice Real time System (TP800 real time fluorescent quantitatives
PCR instrument, TaKaRa) and SYBR Premix Ex Taq (Perfect Real Time) kit (TaKaRa
Biotechnology Inc.Dalian, China)
QPCR identifies that primer is：
IRTKS-F：TTGGCAGGCTACGTCTACCT(SEQ ID NO：11)
IRTKS-R：GCGCCTAGGACTTGAGACTG(SEQ ID NO：12)
Actin-F：AATCGTGCGTGACATTAAGGAG(SEQ ID NO：13)
Actin-R：ACTGTGTTGGCGTACAGGTCTT(SEQ ID NO：14)
PCR reaction systems (25 μ l)：The 2 μ l of μ l, cDNA of 2XSYBR Premix Ex Taq 12.5,10 μM of forward and reverse primers
0.5 μ l, sterile purified water supplies cumulative volume to 25 μ l.
PCR reaction conditions (two step method amplification)：95℃10s；95 DEG C of 5s, 60 DEG C of 30s, totally 38 circulations.
The PCR primer that amplification is obtained carries out solubility curve analysis, and the response procedures of solubility curve generation are 95 DEG C of 15s, 60
DEG C 30s, 95 DEG C of 15s, Dissociation time 4s.
The reaction result of quantitative fluorescent PCR is shown in Fig. 5, as shown in Figure 5 in white adipose, kidney, spleen, lung, IRTKS in pancreas
The expression of mRNA is consistent with WT, and the mRNA of IRTKS is not expressed in LKO mouse livers.
3rd, the extraction of mouse liver histone and immunoblotting assay
Prepare the protein lysate containing protease and inhibitors of phosphatases in advance：20mM Tris pH8.0,137mM
NaCl, 10% glycerine, 1%NP-40 (Nonidet P40), 2mM EDTA, protease and inhibitors of phosphatases.It is placed in ice
On, ground tissue is put into 20min in lysate, 4 DEG C, 120000rpm/min centrifugations 20min.Draw supernatant, albumen
BCA is quantified, immunoblotting assay, and anti-IRTKS (resists) more rabbit, anti-Actin (Santa Cruz).Immunoblot results are shown in
It is consistent with the result of quantitative fluorescent PCR that Fig. 6, Western-blot are verified.
Analyzed and identified by above-mentioned, the IRTKS genes liver specificity for demonstrating the structure of embodiment 1 rejects mouse model
Reliability.
Embodiment 3, wild type and liver specificity reject the glycometabolism correlation analysis of IRTKS mouse
1st, 5.5 monthly age male mice body weight detection finds that liver specificity rejects IRTKS Mouse Weights apparently higher than wild
Type Mouse Weight is shown in Fig. 7.
2nd, IRTKS-LKO and WT mouse fasting blood sugars are detected with Roche blood glucose meter (Roche Accu-Chek), is as a result seen
Fig. 8.IRTKS-LKO mouse blood sugars are significantly higher than WT.
3rd, the male mice at 5.5 monthly ages, overnight fasting, with the glucose of 1ml syringe intraperitoneal injections 1.5g/kg, difference
Every blood glucose value of mouse is determined after (0) and injection within 15,30,60,90,120 minutes, calculate each time point before the injection
The average value and standard deviation of mouse blood sugar, and carry out student ' s t-test statistical analyses, with time point as abscissa, blood sugar
It is ordinate to be worth, and standard deviation is error amount, draws glucose tolerance curve, as a result sees Fig. 9.
4th, 5.5 monthly age male mice liver tissue slices, HE coloration results are shown in Figure 10.Occur in LKO mouse liver tissues
More vacuole sample fat becomes.
Complex chart 7-10 results are visible, and exception occurs in IRTKS gene liver organization specific depletion mouse glycometabolism, has
The features such as hyperglycaemia, glucose-tolerant.
Specific embodiment of the invention is described above.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can within the scope of the claims make various deformations or amendments, this not shadow
Sound substance of the invention.
SEQUENCE LISTING
<110>Shanghai Communications University
<120>Mouse model and build and application that IRTKS genes liver specificity is rejected
<130> DAG28026
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer P1 IRTKS-5P111
<400> 1
tctagacaat gtgcttgcag cc 22
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer P2 neo-reverse
<400> 2
ggcctacccg cttccattgc tc 22
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer P3 neo-Lh
<400> 3
ccgtgccttc cttgaccctg g 21
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer P4 IRTKS-3P211
<400> 4
aactcagacg cagaaaccag tc 22
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer P5
<400> 5
tagttgccag ccatctgttg 20
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer P6
<400> 6
catcctgcac tgctcctgta 20
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer P7
<400> 7
ctggggttcc tgagactttg 20
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer P8
<400> 8
catcctgcac tgctcctgta 20
<210> 9
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer P9
<400> 9
aacgctggtt agcaccgcag g 21
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer P10
<400> 10
tatctcgcgc ggctccgaca 20
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer I RTKS-F
<400> 11
ttggcaggct acgtctacct 20
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer I RTKS-R
<400> 12
gcgcctagga cttgagactg 20
<210> 13
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer Actin-F
<400> 13
aatcgtgcgt gacattaagg ag 22
<210> 14
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer Actin-R
<400> 14
actgtgttgg cgtacaggtc tt 22
Claims (10)
1. the construction method of the mouse model that a kind of IRTKS genes liver specificity is rejected, it is characterised in that by homologous recombination
Method, contain in instead of mouse IRTKS genes with 2.245-kb frt-neo-frt-loxp-Flox-loxp sequences
The sequence of exon2, obtains IRTKSflox/+Allophenic mice；The allophenic mice that will be obtained is with liver specific expression Cre's
Mouse Alb-Cre mates, final to obtain the mouse model that IRTKS genes liver specificity is rejected.
2. construction method as claimed in claim 1, it is characterised in that the construction method is comprised the following specific steps that：
S1, structure IRTKS-CKO plasmids；
S2, ES cytogene are practiced shooting and screening positive clone；
S3, the positive ES clone's injection Mouse Blastocysts, obtain IRTKSflox/+Allophenic mice；
S4, chimera hero mouse and raettin are bred, obtain the positive F1 generation mouse of both arms；
S5, F1 generation mouse or its offspring are mated with Alb-Cre mouse.
3. construction method as claimed in claim 2, it is characterised in that in step S1, IRTKS-CKO DNAs NotI enzymes
Cut, isolate and purify, absolute ethyl alcohol precipitation, aseptic PBS is resuspended, obtains IRTKS-CKO plasmids.
4. construction method as claimed in claim 2, it is characterised in that in step S2, ES cells are SCR012, colony screening bar
Part is 300 μ g/ml G418 and 2 μM of GanC are screened 8 days.
5. construction method as claimed in claim 2, it is characterised in that step S2 also includes identifying positive ES grams with PCR method
It is grand；The primer pair sequence such as SEQ ID NO of 5 ' PCR identifications：1st, shown in 2；The primer pair sequence such as SEQ ID NO of 3 ' PCR identifications：
3rd, shown in 4.
6. construction method as claimed in claim 2, it is characterised in that in step S3, the allophenic mice is that chimeric rate exists
More than 50% male mice.
7. construction method as claimed in claim 2, it is characterised in that during the mouse and step S4 of blastaea be provided in step S3
Raettin is C57BL systems mouse.
8. construction method as claimed in claim 2, it is characterised in that step S5 also includes identifying genotype with PCR method；It is wild
Raw type WT PCR's identifies that primer pair is SEQ ID NO：7th, 8, saltant type Neo PCR's identifies that primer pair is SEQ ID NO：5、
6。
9. the IRTKS genes liver specificity that a kind of construction method as described in any one in claim 1~8 is obtained is rejected
Mouse model.
10. the IRTKS gene liver specificities that a kind of construction method as described in any one in claim 1~8 is obtained are picked
Application of the mouse model for removing in preparing or screening Remedies for diabetes.
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| CN110777203A (en) * | 2018-12-29 | 2020-02-11 | 湖南师范大学 | Application of Kctd10 gene in treatment of liver diseases |
| CN111206054A (en) * | 2020-02-14 | 2020-05-29 | 河北医科大学第三医院 | Construction method of liver HO-1 gene conditional knockout animal model by using CRISPR-Cas9 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107446950A (en) * | 2017-08-08 | 2017-12-08 | 西北工业大学 | MACF1 pinpoints transgene mouse model construction method |
| CN109355309A (en) * | 2018-10-18 | 2019-02-19 | 江苏集萃药康生物科技有限公司 | A kind of construction method of CD3E gene modification humanized animal's model |
| CN109355309B (en) * | 2018-10-18 | 2021-02-05 | 江苏集萃药康生物科技股份有限公司 | Construction method of CD3E gene modified humanized animal model |
| CN110777203A (en) * | 2018-12-29 | 2020-02-11 | 湖南师范大学 | Application of Kctd10 gene in treatment of liver diseases |
| CN111206054A (en) * | 2020-02-14 | 2020-05-29 | 河北医科大学第三医院 | Construction method of liver HO-1 gene conditional knockout animal model by using CRISPR-Cas9 |
| CN111206054B (en) * | 2020-02-14 | 2022-12-30 | 河北医科大学第三医院 | Construction method of animal model for conditionally knocking out liver HO-1 gene by using CRISPR-Cas9 |
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