CN104131036B - Construction method and application of conditional ivermectin receptor IVMR transgenic mouse model - Google Patents
Construction method and application of conditional ivermectin receptor IVMR transgenic mouse model Download PDFInfo
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Abstract
The invention relates to a construction method and an application of a conditional ivermectin receptor (IVMR) transgenic mouse model, and belongs to the technical fields of conditional animal model construction method and application. Under the action of a Cre recombinase, all cells (especially cerebral neurons) of the mouse model can conditionally express the ivermectin receptor IVMR, and the excitability of the neurons expressing the ivermectin receptor IVMR can be down-regulated by applying a drug ivermectin (IVM). Compared with the prior art, the technical method (including a drug administration mode, and targetedly using a transgenic mouse for specific expression of Cre) does not has any brain damage on the mouse. Influence of the used drug ivermectin IVM on animals has a sufficiently long half-life period, and animal behavioral experiments are suitably carried out. In addition, the half-life period is relatively stable, the experiments can be again carried out after drug failure, and before-and-after comparison experiments are conveniently carried out on the same animal.
Description
Technical field
The present invention relates to the construction method of conditional transgenic animal model and application, especially relate to a kind of conditional she
The construction method of dimension rhzomorph receptor IVMR transgene mouse model and the application in neuronal function research.
Background technology
One of Neuroscience Research field key issue is exactly activity (irritability) and the behavior of specific neuron
Cause effect relation.To this problem, have been developed that much new method at present in the world, such as, the molecule behaviour of synapse transmission
Make the ion channel method of method, light genetic method and part gate.Particularly have been developed in and led to the ion of part gate
The method that road is used for inducible and reversible regulation and control neuronal activity.Such as, fruit bat Allatostatin receptor is in quilt
In the case of Allatostatin activation, can be with the generation of the action potential of inhibitory neuron, this method is moved in suckling
Begin to use in thing brain.However, this molecule of Allatostatin can not be through blood brain barrier it is necessary to by invasive method
(such as brain punching pipe laying) is injected in brain, and this just seems very inconvenient when carrying out extensive animal behavior experiment.
Recently, a kind of GABA of through engineering approachesAReceptor has been used for the specific brain neuron of silence, and feature is to drug zolpidem
(zolpidem) insensitive.But this system also has obvious defect it is simply that GABA must expressedAγ 2 subunit
Work in neuron.The system that the third can reduce neuron granting action potential is exactly the chlorine of ivermectin IVM gate
Ion channel (this passage derives from nematicide glutamic acid chloride channel GluCl α and β subunit), has been reported this passage of display
(Lerchner, the W.et al.2007.Reversible silencing of that works is can be very good in mouse brain
Neuronal excitability in behaving mice by a genetically targeted, ivermectin-
Gated Cl-channel.Neuron 54,35-49.).But, a big restriction of this system is exactly to need express alpha simultaneously
With two subunits of β.In order to overcome this problem, glycine α 1 receptor in people source has been carried out F207A and A288G by Lynagh et al.
Two point mutation, make this receptor no longer sensitive to glycine, and (people glycine α this mutation after sensitive to ivermectin IVM
1 receptor is named as ivermectin receptor IVMR), which provides a kind of can induce well and reversible silence neuron
Instrument (Lynagh, T.＆Lynch, J.W.2010.An Improved Ivermectin-activated Chloride
Channel Receptor for Inhibiting Electrical Activity in Defined Neuronal
Populations.J Bio Chem 285,14890-14897).But so far, only construction expression ivermectin in the world
The report that the virus of receptor IVMR receptor is studied, and be prepared as transgene mouse model and have not been reported.
Content of the invention
The purpose of the present invention is exactly for the deficiency for existing shortage ivermectin receptor IVMR transgenic mice, and carries
For a kind of impact to animal behavior for activity that can preferably be used for neuron research conditional (or claiming induction type) she
Dimension rhzomorph receptor IVMR transgene mouse model and its construction method and application.
By the sequence of IVMR be integrally inserted into Rosa26 gene locis downstream (this gene be an expression extensively and water
Flat very strong gene), after the STOP element that a loxP site surrounds.Then such plasmid electricity is rotated into mice
Carry out homologous recombination, positive-negative selection, multiple PCR carry out screening the embryonic stem cell completing homologous recombination in embryonic stem cell.Again
Positive embryo stem cell transplantation is entered in the blastaea of dams, after choosing birth, is fitted together to horizontal highest gomphosis mouse and wild type
Mice carries out copulation, and the generation mice being accredited as the positive is IVMR transgenic mice.This mice can be entered with Cre recombinase mice
Row copulation or Naoliqing capsule injection Cre expression virus, so in the presence of specific Cre recombinase, before IVMR
STOP sequence be cut away, IVMR is able to express in specific neuron.IVMR transgenic mice abdomen in Cre expression
Chamber administration IVM can activate IVMR, reduces the activity of neuron.
Therefore, it is an object of the present invention to provide a kind of conditional expresses the transgenic mice of ivermectin receptor IVMR
Model.
It is a further object to provide the transgenic that a kind of structure conditional expresses ivermectin receptor IVMR is little
The method of mouse model, the method comprises the following steps：
(1) sequence of IVMR-2A-GFP is inserted into the downstream of Rosa26 gene locis, wraps immediately preceding a loxP site
After the STOP element enclosing, build Rosa26-IVMR targeting vector；
(2) Rosa26-IVMR targeting vector electricity is rotated into and in mouse embryo stem cell, carry out homologous recombination, through positive and negative sieve
Choosing and PCR screen the embryonic stem cell completing homologous recombination；
(3) positive embryo stem cell transplantation is entered in the blastaea of dams, the gomphosis mouse choosing birth is little with wild type
Mus carry out copulation, and the generation mice being accredited as the positive is IVMR transgenic mice；
(4) virus of expression Cre recombinase is entered in the brain of IVMR transgenic mice through stereotaxical injection, or by spy
The mice determining neuron expression Cre recombinase is copulationed with IVMR transgenic mice, the double positive generation mices obtaining, and becomes
Work(builds conditional IVMR transgenic mice；
(5) ivermectin is administered to the IVMR transgenic mice of injecting virus or specific neuron expression Cre recombinase
IVM, reduces the activity of specific neuron.
Above-mentioned steps (2) need to use the positive mouse embryo stem cell of gene-specific primer evaluation and screening, step (3)
(4) need to use the positive IVMR and Cre transgenic mice of gene-specific primer identification.
It is also another object of the present invention to provide this mouse model study specific neuron activity and animal behavior it
Between relation application.
The conditional that the present invention provides expresses the model of transgenic mice and the conventional art phase of ivermectin receptor IVMR
The having the beneficial effect that of ratio：
First, compared with the technology of existing construction expression ivermectin receptor IVMR virus, the present invention is built into first
Work(ivermectin receptor IVMR transgenic mice, has no in the world at present and has been reported that.
Secondly, compared with remaining can reduce the technical method of neuronal activity, particularly now especially popular in the world
Light genetics technology (need brain pipe laying insert optical fiber) compare, the technical method of the present invention (include administering mode, for
Transgenic mice using particular expression Cre) there is no any brain injury to mice.
Finally, the impact to animal for the medicine ivermectin IVM that the present invention uses has the sufficiently long half-life, be suitable for into
Action thing behavioral experiment.In addition the half-life is also relatively stable, can test again after drug failure, easily to same dynamic
Comparative experimentss before and after the carrying out of thing.
Brief description
Fig. 1 is the schematic diagram building the gene targeting strategy that IVMR transgenic mice uses；
Fig. 2 identifies for PCR and homologous recombination mouse embryo stem cell occurs；
Fig. 3 be obtain IVMR transgenic mice after make IVMR gene expression, neuronal activity decline technical step and plan
Slightly；
Fig. 4 is that to be expelled to IVMR transgenic by the method that Naoliqing capsule is injected little by the virus of expression Cre recombinase
The expression of IVMR after the brain of Mus, is identified using the RNA in situ hybridization of IVMR gene；
Fig. 5 is will to express the transgenic of specific Cre recombinase (CamKII-Cre of CamKII promoters driven Cre expression)
Mice is copulationed with IVMR transgenic mice, obtains the CamKII-Cre of double positives；IVMR transgenic mice, using IVMR gene
RNA in situ hybridization identifies the expression of IVMR.
Specific embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment：
1st, build the targeting vector of IVMR transgenic mice
The plasmid comprising IVMR-2A-GFP gene order teaches favour by University of Queensland Joseph W.Lynch
Give, Rosa26-CAG targeting vector is buied from U.S. Addgene website.Using general molecular clone method by IVMR-2A-
GFP gene order (GFP is green fluorescent protein, and 2A is from cleavage sequence, conveniently by IVMR and GFP albumen separately) is inserted into
After the STOP site of Rosa26 targeting vector, replace the tdTomato sequence in initial carrier, thus successfully construct comprising
The targeting vector of IVMR gene, as shown in Figure 1B, the 5 ' homology arms (5 ' arm) used in targeting vector (B) and 3 ' homology arms (3 '
Arm) it is located between first and second exons (exon) of wild type Rosa26 gene locis (A).Homologous recombination carries practicing shooting
Gene order between two homology arms of body is inserted between the homologous sequence of embryonic stem cell Rosa26 gene locis, including
STOP site, IVMR-2A-GFP expressed sequence and multiple selection markers that CAG promoter, loxp surround etc..Wherein, 5 ' homology
Arm and 3 ' homology arms identification primer are also marked on Fig. 1 C.Wherein, this targeting vector contains 5 ' homology arms of 1.1kb length
3 ' the homology arms (3 ' arm) of (5 ' arm) and 4.3kb length, CAG promoter, the STOP sequence of loxp encirclement, IVMR-2A-GFP table
Reach sequence and PGK-NeoR the and PGK-DTA-pA sequence for screening and cloning.
2nd, homologous recombination
Then by after the targeting vector building linearisation, 129/B6 mouse hybrid is gone to by Amaxa electroporation electricity
In the embryonic stem cell line G442 of a generation.The embryonic stem cell that homologous recombination occurs can express Neo resistant gene, therefore, it is possible to
Culture fluid containing antibiotic G418 survives；And PGK-DTA-pA sequence will not be integrated into dry thin by homologous recombination
In the genome of born of the same parents, so stem cell will not be damaged by the toxicity of DTA.By the embryonic stem cell of above-mentioned positive-negative selection, enter
Their total genomic dna of onestep extraction, carries out the PCR identification of homology arm.Under the primer sites of 5 ' and 3 ' homology arms such as Fig. 1 C
Face 5 ' arm-F/R, shown in 3 ' arm-F/R, primer sequence is as follows：
5’arm-F：5 '-CGCGGAACTCCATATATGGGCTATG-3 ', such as SEQ ID NO：Shown in 1；
5’arm-R：5 '-GGCTCCTCAGAGAGCCTCGGCTAG-3 ', such as SEQ ID NO：Shown in 2；
3’arm-F：5 '-AGACCATTCTCAGTGGCTCAACAAC-3 ', such as SEQ ID NO：Shown in 3；
3’arm-R：5 '-ATTCGGCTATGACTGGGCACAACA-3 ', such as SEQ ID NO：Shown in 4；
The result of PCR identification is as shown in Fig. 2 the embryonic stem cell that 5 ' and 3 ' homology arm PCR are the positive is 2,4,5,7 and
No. 10 clones.This shows, this five clones are the embryonic stem cells through homologous recombination.
3rd, blastaea injection and chimera obtain
4,5 and No. 10 Embryonic stem cell clones that selection 5 ' and 3 ' homology arm PCR are the positive are expelled to C57BL/ respectively
In the blastaea of 6J dams.Thereafter these other embryonic stem cells in the embryonic stem cell and blastaea of homologous recombination can be integrated
Together, develop into embryo to be born until smooth.Because the fur of 129 Strains of Mouses is white, the fur of C57BL/6J strain
It is black, along with 4,5, No. 10 Embryonic stem cell clones using are all above-mentioned 2 strains mixing, so the mice of birth
Fur be in chequered with black and white, referred to as allophenic mice.By allophenic mice and C57BL/6J Strains of Mouse phase copulation, little in filial generation
Mus enter performing PCR with following primer, and the mice being accredited as the positive is ivermectin receptor IVMR transgenic mice.
WT-F：5 '-TGCATAAAACCCCAGATGACTACC-3 ', such as SEQ ID NO：Shown in 5；
WT-R：5 '-GCAATACCTTTCTGGGAGTTCTCT-3 ', such as SEQ ID NO：Shown in 6；
IVMR-F：5 '-TCGACCATGGTAATAGCGATGAC-3 ', such as SEQ ID NO：Shown in 7；
It is simultaneously introduced above 3 primers, PCR program setting is when entering performing PCR identification：94 DEG C of denaturations 5 minutes, 94 DEG C 30
Second, 58 DEG C 30 seconds, 72 DEG C 30 seconds, totally 35 circulation, then 72 DEG C extend 5 minutes, 12 DEG C preservation.The band of wild-type mice is
304bp, and the band of IVMR transgenic mice is 514bp.
To this step, successfully construct IVMR transgenic mice, but in this mice, before IVMR gene also
There is a STOP sequence, so ivermectin receptor IVMR can't express.
4th, the structure of conditional ivermectin receptor IVMR transgenic mice
Can there are following two strategies (as shown in Figure 3) building conditional ivermectin receptor IVMR transgenic mice：
A, using Cre restructuring expression of enzymes virus (including adeno-associated viruses AAV, slow viruss Lentivirus etc.)；B, using specific Cre weight
Group enzyme transgenic mice (expresses the transgenic mice of Cre recombinase) with specific promoter sequence.Specifically, when using Cre
During restructuring expression of enzymes virus (Fig. 3 A), the head of postanesthetic IVMR transgenic mice is fixed on stereotaxic instrument, cuts off head
Skin exposes skull, with relevant position (as striatum) punching on skull for the microbit, using capillary glass tube, AAV is viral
It is injected in striatum.Another kind of method (Fig. 3 B) is exactly (as CamKII promoter is driven by specific Cre recombinase transgenic mice
The transgenic mice of dynamic Cre expression) copulation with IVMR transgenic mice, in filial generation, Cre and IVMR is screened by PCR double positive
Mice be Cre；IVMR transgenic mice.
Screen the WT-F that the positive primer of IVMR and PCR program are used with previous step, WT-R, IVMR-F are identical.
The positive primer of screening Cre is as follows：
Cre-F：5 '-TCGATGCAACGAGTGATGAG-3 ', such as SEQ ID NO：Shown in 8；
Cre-R：5 '-TCCATGAGTGAACGAACCTG-3 ', such as SEQ ID NO：Shown in 9；
PCR program setting is 94 DEG C of denaturations 5 minutes, 94 DEG C 30 seconds, 60 DEG C 30 seconds, 72 DEG C 30 seconds, totally 30 circulations, so
72 DEG C extend 5 minutes afterwards, 12 DEG C of preservations.Wild-type mice no band, the band of Cre positive mice is about 400bp.
Whether pass through expressing viral Cre recombinase or express Cre by copulationing with Cre recombinase transgenic animal,
It is provided to cut off the STOP sequence before IVMR gene order, so that this ivermectin receptor IVMR smoothly gives expression to
Come.So have successfully been obtained conditional ivermectin receptor IVMR transgenic mice.
Finally ivermectin IVM is passed through lumbar injection little to the IVMR transgenic injecting Cre expression virus in brain
Mus and Cre；In IVMR transgenic mice, ivermectin IVM activates ivermectin receptor IVMR in brain, makes the work of neuron
Property decline.
5th, the identification of conditional ivermectin receptor IVMR transgenic mice
Carry out the identification of the conditional ivermectin receptor IVMR transgenic mice that two kinds of strategies obtain.The RNA of gene
In situ hybridization is a kind of method of conventional detection gene mRNA expression.The AAV of injection expression Cre in IVMR transgenic mice
Virus, after 7 days, mouse anesthesia is put to death, and is successively irrigated with PBS and 4%PFA, fixing after 4%PFA, and frozen section is carried out in situ
Hybridization, royal purple chrominance signal is the positive signal of hybridization, shows that this cell expresses the mRNA (Fig. 4 B) of ivermectin receptor IVMR,
And do not inject the transgenic mice (Fig. 4 A) of Cre virus or the wild-type mice (Fig. 4 C) of injection Cre virus, all there is no IVMR
Expression.In addition, hybridize CamKII-Cre mice with IVMR transgenic mice, obtain double positive progeny mice CamKII-
Cre；IVMR transgenic mice, this mice is put to death, irrigates, is cut into slices with above-mentioned similar method, carries out the RNA of IVMR gene
In situ hybridization.The positive signal all having in situ hybridization in Hippocampus and cerebral cortex extensive region is expressed, and this shows in CamKII-
Cre；In Hippocampus and cerebral cortex in IVMR transgenic mice mouse brain, all there is stronger IVMR gene expression, as Fig. 5 institute
Show, CA1 in Fig. 5：Hippocampal CA 1；DG：Hippocampal dentate；I-VI：Corticocerebral layering.
The above-mentioned description to embodiment is to be understood that and use invention for ease of those skilled in the art.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiment without through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, ability
, according to the announcement of the present invention, the improvement made without departing from scope and modification all should be in the present invention for field technique personnel
Protection domain within.
Claims (4)
1. a kind of construction method of conditional ivermectin receptor IVMR transgene mouse model is it is characterised in that include following
Step：
(1) sequence of IVMR-2A-GFP is inserted into the downstream of Rosa26 gene locis, surrounds immediately preceding a loxP site
After STOP element, build Rosa26-IVMR targeting vector；
(2) Rosa26-IVMR targeting vector electricity is rotated into and in mouse embryo stem cell, carries out homologous recombination, through positive-negative selection and
PCR screening completes the embryonic stem cell of homologous recombination；
(3) positive embryo stem cell transplantation is entered in the blastaea of dams, the gomphosis mouse choosing birth is entered with wild-type mice
Row copulation, the generation mice being accredited as the positive is IVMR transgenic mice；
(4) virus of expression Cre recombinase is entered in the brain of IVMR transgenic mice through stereotaxical injection, or by specific god
Mice through unit's expression Cre recombinase is copulationed with IVMR transgenic mice, the double positive generation mices obtaining, successful structure
Build conditional IVMR transgenic mice.
2. the construction method of a kind of conditional ivermectin receptor IVMR transgene mouse model according to claim 1,
It is characterized in that, further include respectively in step (3)～step (4) to utilize specific gene amplimer to the mice being obtained
Carry out genotype identification.
3. a kind of conditional ivermectin receptor IVMR transgene mouse model being obtained using claim 1 method is in research brain
Neuronal activity declines, and the application in the relation between neuronal function and animal behavior.
4. application according to claim 3 is it is characterised in that recombinate to injecting virus or specific neuron expression Cre
The IVMR transgenic mice administration ivermectin IVM of enzyme, reduces the activity of specific neuron.
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