CN108118069A - New people's induced pluripotent stem cells system of simulation Alzheimer disease and application thereof - Google Patents
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Abstract
The present invention relates to a kind of methods for the cell model for preparing Alzheimer disease (AD), it includes AD related genes being integrated into hiPSC the horizontal increase of the beta-secretase of hiPSC and/or 42 peptides of A β is induced to increase, and also relates to the cell model of the Alzheimer disease (AD) prepared by this method.
Description
Technical field
The present invention relates to biomedical sectors, and the relevant cell of physiology is created especially for Alzheimer disease (AD)
Model.In particular it relates to prepare AD cell models by genetic modification people induced pluripotent stem cells (hiPSC)
Method.
Background technology
Alzheimer disease (AD) is chronic neurodegenerative disease, is the most common reason of dementia.Based on twins
With the Review Study of family, the gene genetic rate scope of Alzheimer disease is from 49% to 79%1-2.About 0.1% disease
Example is autosome (non-gunther sex-linked) dominant inheritance of familial, is fallen ill before 65 years old3.The disease of this form claims
For early onset family Alzheimer disease.Most of autosomal dominant familial AD are attributable to one of following three kinds of genes
Mutation：Those genes of encoding amyloid precursor albumen (APP) and presenilin 1 and 24.In APP and presenilin genes
Most of mutation increases the generation for the little albumen matter for being known as A β 42, and A β 42 are the main components of senile plaque expelling5。
Most of cases of Alzheimer disease do not appear as autosome-dominant inheritance, are known as sporadic AD,
Middle environment and hereditary difference may be risk factor.Most significant genetic risk factors are 4 allele of ε of apo E
Hereditary (APOE)6-7.At least a kind of 4 allele of APOE ε of 40% to 80% people with AD7.4 allele of APOE ε
The risk of disease in heterozygote is improved three times, 15 times are improved in homozygote3。
TREM2 gene mutations are related to the Alzheimer disease occurrence risk for being higher by 3 to 5 times8-9.The effect commonly approved
Mechanism is when TREM2 is mutated, and the leucocyte in brain is no longer able to the amount of control amyloid beta.
There are two different features for AD tools：One is the presence of the extracellular plaque containing amyloid beta-albumen (A β),
The other is cellular neurofibrillary tangles (NFT).Although the true pathology of AD is still unclear, many decades research
It accumulates and provides strong evidence for amyloid cascade hypothesis.According to this it is assumed that causing the critical event of AD seemingly
Specific peptide, wherein (the A β of amyloid beta peptide 42 are formed in the process of amyloid precusor protein (APP)42) it is to be easy to
Aggregation forms A β42Oligomer and the sticky peptide for subsequently forming amyloid patch, this is first feature of AD.Aβ42Aggregation
The presence of body and amyloid patch can trigger the inflammation of neuron, and increase the expression of calcium-activated kinases, anti-
Come over to induce the Hyperphosphorylationof of Protein tau, Protein tau stablizes the cytoskeletal microtubule in neuron.The super phosphorus of Protein tau
Acidifying causes the formation of neurofibrillary tangles in neuron, i.e. the second of AD feature, and it is dead to subsequently result in neuron
It dies10-14。
Three kinds of enzymes, alpha-secretase enzyme, beta-secretase and gamma-secretase participate in the cutting of APP.In normal processes, APP is first
First cut by alpha-secretase enzyme or beta-secretase, and the product further cut is processed into different length by gamma-secretase
The mixture of peptide.If APP is cut by beta-secretase, product will further be cut by gamma-secretase, to generate solubility
40 amino acid 4 amyloid (A β40) or 42 amino acid peptide (A β42), the peptide aggregation of 42 amino acid is together
Insoluble aggregates are formed, therefore form amyloid plaques.The strong evidence of amyloid cascade hypothesis comes from familial
The research of AD (FAD), the APP or presenilin (PS) gene for showing all familial AD patients have mutation.App gene
Mutation causes abnormal APP albumen, and the abnormal APP albumen preferentially generates more A β peptides by beta-secretase cutting；And PS
Gene mutation causes preferentially to generate A β42Peptide, A β42Peptide is the ingredient of amyloid patch.Another important observed result
It is that the expression of beta-secretase and enzymatic activity significantly raise in AD patient, is particularly representing the sporadic of more than 90% AD groups
In AD (SAD) patient.In the brain area domain influenced by amyloid beta deposition beta-secretase albumen and activity level rise and
This rise is always maintained at, but the neuron and cynapse in AD are substantially lost15-17。
Beta-secretase 1 (BACE1), also referred to as β-site amyloid precusor protein nickase 1, β sites APP nickases 1,
Film associated aspartate protease 2, memapsin-2, aspartyl protease 2 and ASP2 are the shapes in the nerve cell of periphery
Important aspartic protease during into myelin18.In people, it is by BACE1 gene codes.It is circumscribed that cell is carried out by BACE1
It cuts APP and generates soluble cell outer segment and the cell membrane binding fragment of referred to as C99.By gamma-secretase in its transmembrane structure
Domain internal cutting C99 discharges the intracellular domain of APP and generates amyloid beta.Since gamma-secretase cuts APP ratios
BACE1 cuts APP closer to cell membrane, therefore gamma-secretase removes the segment of amyloid-β peptides.Pass through alpha-secretase enzyme
Rather than BACE1 tentatively cuts APP and can prevent from finally generating amyloid beta.
Different from APP and presenilin (PS) albumen, the gene mutation without known coding BACE1 causes early onset man
Race's property Alzheimer disease, this is rare disease form.However, have shown that the horizontal in more conventional evening hair of this enzyme
It is raised in the sporadic Alzheimer's disease of property.The purpose of physiology of BACE cutting APP and other transmembrane proteins is unknown.
BACE2 is the homologue for being closely with BACE1, without the report of internal APP cuttings.
Since the generation of A β -42 peptides plays a major role in the formation of amyloid patch, a past 20 years left side
Right all drug discovery efforts are all concentrated on reduces A β -42 by inhibiting beta-secretase activity or inhibiting A β -42 aggregations
Peptide19-26.However, one of main problem of AD drug developments is a lack of suitable AD models.AD models foremost so far
It is to be generated by the way that APP the and PS1 genes of mutation are inserted into mouse genome with showing AD phenotypes in aged mouse
5xFAD transgenic mices27.Although 5xFAD mouse models widely use, have in drug development in its effectiveness
Two major defects.First, the central nervous system of people and the central nervous system of mouse are very different, therefore in 5xFAD
The drug candidate tested on model is poor generally for the predictability of its influence to people.Second, 5xFAD mouse need 6 to 8
A month or it is longer can just show phenotype, therefore the model is obviously not suitable for early screening AD drug candidates.
Pluripotent stem cell has very big development prospect in terms of cell model is prepared, because they can be infinitely numerous
It grows, and generates other cell types in human body, such as neuron, heart, pancreatic cell and liver cell.People induces pluripotency
Stem cell (hiPSC) is a kind of pluripotent stem cell, can directly be generated from adult cell.IPSC technologies are by Shinya
What Yamanaka was started in the laboratory of kyoto, Japan, he is open in 2006, and introduce encoding transcription factors four are specific
Adult cell can be converted into pluripotent stem cell by gene.This disruptive technology is reprogramming of somatic cells technology, makes researcher
Terminally differentiated cells such as skin fibroblasts can be converted back into the embryonic stem cell stage28-29。
HiPSC can be obtained from the patient with various diseases and Immortalization, therefore provides unlimited cell and come
Source.Importantly, these hiPSC from patient can be divided into the relevant primary cell of disease again, and in cellular water
Pingxian shows disease phenotype30-36.This forms to create a kind of common strategy of the cell disease models based on hiPSC.The strategy
It is to generate hiPSC from the patient with disease that clarifies a diagnosis, the hiPSC from patient is then divided into target cell type,
To show disease phenotype in vitro.
In addition to the hiPSC from patient, researcher is attempting to establish another strategy of cell disease models always.
They generate hiPSC from non-diseased person, and Disease-causing gene then is introduced hiPSC by genome editing technique.
In order to generate suitable AD cell models from hiPSC, it is necessary to meet following standard：It must be in a physiologically
It is equal to people's functional nerve member；It must show AD phenotypes in vitro within the relatively short time；And it allows for consistent
Ground reproduce largely with the relevant human neures of AD.Although by the research of many decades, this is not really prepared successfully so far
Kind cell model.
The content of the invention
It is at least some above-mentioned to meet that the present invention prepares the method for the cell model of Alzheimer disease (AD) by offer
Demand, the described method includes genetic modification hiPSC to generate the higher water compared with the grade genes hiPSC for deriving cell model
Flat beta-secretase and β -42 peptides.
For the present invention, AD related genes are introduced non-lesion hiPSC, obtain isogenic line, i.e., with phase by the present inventor
The same sick cell system of genetic background and non-lesion cell line, and due to the AD dependency basis of genome editing technique introducing
The pile up effect of cause, sick cell tie up in the relatively short time and show AD phenotypes in cellular level in vitro.
In one aspect, the method that the present invention provides the cell model for generating Alzheimer disease (AD), including by AD
Related gene integrates to induce beta-secretase horizontal with hiPSC and/or A β -42 peptides increase.In one embodiment, it is described
AD related genes are overexpressed in hiPSC for composition.In one embodiment, the AD related genes are that AD is caused to send out
Mutation APP or the PS gene of disease, particularly PS1dE9 genes.In one embodiment, the AD related genes are BACE1
Gene.In one embodiment, the AD related genes be selected from cause AD fall ill mutation APP, PS1dE9 gene and
One or more in BACE1 genes.
In some embodiments, the AD related genes are integrated by way of locus specificity in hiPSC.It is excellent
Selection of land, the AD related genes are in AAVS1 integrations into hiPSC.
In one aspect, the present invention provides the cell model of the Alzheimer disease (AD) generated by the above method.
In one aspect, the present invention include modification hiPSC method, include importing AD related genes to hiPSC so that
Its composition is overexpressed.In one embodiment, the AD related genes are directed into hiPSC, the table by expression vector
The nucleotide sequence of coding AD GAP-associated protein GAPs and reporter molecule is included up to carrier, nucleotide sequence is operable with promoter
Ground connects, and the promoter is for driving the promoter of the high level gene expression in mammalian expression vector.At one
In embodiment, the promoter is PGK promoters or CAG promoters.In one embodiment, will be controlled by promoter
Drug screening gene comprising coding AD GAP-associated protein GAPs and reporter molecule nucleotide sequence identical expression vector in or
It is further incorporated into individual expression vector in hiPSC, the promoter is for driving in mammalian expression vector
The promoter of high level gene expression.In one embodiment, the promoter is PGA promoters or CAG promoters.
In one embodiment, expression vector further includes the nucleotide sequence for site-specific integration, it is preferable that with people
The homologous nucleotide sequence in AAVS1 sites.
In one aspect, the present invention provides expression vector, and it includes coding AD GAP-associated protein GAPs and the nucleotide of reporter molecule
Sequence, nucleotide sequence are operably connected with promoter, and the promoter is for driving mammalian expression vector
In high level gene expression promoter.In one embodiment, the promoter starts for PGK-1 promoters or CAG
Son.In one embodiment, the carrier further includes the nucleotide for the drug screening gene that coding is controlled by promoter
Sequence, the promoter are for driving the promoter of the high level gene expression in mammalian expression vector.In a reality
It applies in mode, the promoter is PGK-1 promoters or CAG promoters.In one embodiment, the carrier is further
Include the nucleotide sequence for site-specific integration, it is preferable that the homologous nucleotide sequence with people AAVS1 sites.One
In a embodiment, drug screening gene be antibiotics resistance gene, it is preferable that puromycin, neomycin, kanamycins or
Genetic resistance genes.In one embodiment, reporter is encoding green fluorescent protein or red fluorescent protein
Gene.In one embodiment, all elements in carrier are all arranged by the order for being conducive to the AD related gene expressions
Row.Preferably, all elements in carrier are ranked sequentially as cis.
In one aspect, the present invention provide genetic constructs, it includes nucleic acid sequence encoding it is as follows：First promoter；
The drug screening gene controlled by the first promoter；Second promoter；It is connected with the reporter controlled by the second promoter
Related gene；The homologous sequence with people AAVS1 sites, wherein all elements are all cis arrangement.In one embodiment,
First promoter is PGK-1 or CAG promoters；The drug screening gene is antibiotics resistance gene；Described second opens
Mover is PGK-1 or CAG promoters；The AD related genes are BACE1 and the reporter is encoding green fluorescent egg
White or red fluorescent protein gene, it is preferable that the gene of encoding green fluorescent protein.In one embodiment, described
One promoter is PGK-1 or CAG promoters；The drug screening gene is antibiotics resistance gene；Second promoter is
PGK-1 or CAG promoters；The AD related genes are PS1dE9 and the reporter is GFP.In one embodiment,
Antibiotics resistance gene is puromycin, neomycin, kanamycins or genetic resistance genes.
In one aspect, the present invention was provided by appointing in any one of above-mentioned expression vector or above-mentioned genetic constructs
A kind of hiPSC cell lines of conversion, in one embodiment, the hiPSC cell lines are used to generate AD cell models.
In one aspect, the present invention provides the hiPSC of the genetic modification as AD cell models, in people AAVS1 sites
It integrates BACE1 genes and composition is overexpressed the BACE1 genes integrated.In one embodiment, with described in no integration
BACE1 genes wait genes hiPSC to compare, and modified hiPSC shows horizontal beta-secretase, A β 42/A β -40 ratios, and/or A
β -42 peptides increase.In one embodiment, modified hiPSC is in people AAVS1 integrations PS1dE9 genes and composition
It is overexpressed the PS1dE9 genes integrated.
In one aspect, the present invention includes the hiPSC of genetic modification for the purposes of high flux screening AD medicines.
In one aspect, the present invention is provided to high flux screening AD therapeutic agent method, including
I) from hiPSC by importing expression vector as described above or genetic constructs as described above preparation A Er to hiPSC
The cell model of Ci Haimo diseases (AD),
Ii) with cell model culture candidate compound two days to two weeks,
Iii) before and after the candidate compound is added in, measurement beta-secretase level, A β -42 concentration and A β 42/
A β -40 compare；
Iv it is) one or more selected from beta-secretase level, A β -42 concentration, A β 42/A β -40 ratios and A β 42/A β -40 ratios
Measured value reduce, represent the candidate compound for treatment AD potential treatment agent.
In one embodiment, the hiPSC comes from people's donor, is preferred from not suffering from the normal human donor of AD, and
And hiPSC is converted by conventional reprogramming method in vitro.In one embodiment, the method is used to screen morning
Phase AD drug.
In one aspect, the present invention provides the drug screening method of screening beta-secretase or A β -42 inhibitor, including
I) BACE1 genes or PS1dE9 genetic modification hiPSC cell lines are overexpressed by composition,
Ii hiPSC cell lines) are divided into functional nerve member again,
Iii) cultivated in the presence of candidate drug compounds functional nerve member and
Iv beta-secretase can be reduced by) measuring horizontal beta-secretase, A β 42/A β -40 ratios, and/or A β -42 concentration and screening
Horizontal, A β 42/A β -40 than, and/or A β -42 concentration compound.
In one embodiment, the described method includes the functional god is cultivated in the presence of candidate drug compounds
Through member up to two days to two weeks.In one embodiment, the hiPSC comes from people's donor, is preferred from not suffering from the normal of AD
People's donor, and hiPSC is converted by conventional reprogramming method in vitro.In one embodiment, pass through to
HiPSC imports expression vector as described above or genetic constructs as described above prepare the hiPSC.
In particular it relates to：
1. a kind of method for the cell model for preparing Alzheimer disease (AD), including AD related genes are integrated into
To induce, the beta-secretase of hiPSC level increases and/or A β -42 peptides increase in hiPSC.
2. 1 method, wherein AD related genes composition in hiPSC is overexpressed.
3. 1 or 2 method, wherein the AD related genes are the mutation APP genes or mutation PS bases that AD is caused to fall ill
Cause.
4. 3 method, wherein the mutation PS genes are PS1dE9 genes.
5. 1 or 2 method, wherein the AD related genes are BACE1 genes.
6. the method for any one of 1-5, wherein the AD related genes be selected from AD is caused to fall ill mutation app gene,
PS1dE9 genes and BACE1 genes.
7. the method for any one of 1-6, wherein the AD related genes are integrated by way of locus specificity
In hiPSC.
8. 7 method, wherein the AD related genes in AAVS1 integrations into hiPSC.
9. the cell model for passing through the Alzheimer disease (AD) that the method for any one of item 1-8 generates.
10. a kind of method for modifying hiPSC, including importing AD related genes to hiPSC so that its composition crosses table
It reaches.
11. 10 method, the AD related genes are directed into hiPSC by expression vector, the expression vector includes
The nucleotide sequence of AD GAP-associated protein GAPs and reporter molecule is encoded, wherein the nucleotide sequence is operably connected with promoter,
The promoter is for driving the promoter of the high level gene expression in mammalian expression vector.
12. 11 method, wherein the promoter is PGK-1 promoters or CAG promoters.
13. 11 or 12 method, by the drug screening gene of promoter control by comprising coding AD GAP-associated protein GAPs and
The identical expression vector of the nucleotide sequence of reporter molecule is further incorporated into the hiPSC by individual expression vector
In, the promoter is for driving the promoter of the high level gene expression in mammalian expression vector.
14. 13 method, wherein the promoter is PGK-1 promoters or CAG promoters.
15. the method for any one of 10-14, wherein the expression vector is further included for site-specific integration
Nucleotide sequence.
16. 15 method, wherein the nucleotides sequence for site-specific integration is classified as and people AAVS1 sites
Homologous nucleotide sequence.
17. a kind of expression vector, it includes coding AD GAP-associated protein GAPs and the nucleotide sequence of reporter molecule, wherein described
Nucleotide sequence is operably connected with promoter, and the promoter is for driving the gene in mammalian expression vector
The promoter of high level expression.
18. 17 carrier, wherein the promoter is PGK-1 promoters or CAG promoters.
19. 17 or 18 carrier, further include coding by promoter control drug screening gene nucleotide
Sequence, the promoter are for driving the promoter of the high level gene expression in mammalian expression vector.
20. 19 carrier, wherein the promoter is PGK-1 promoters or CAG promoters.
21. the carrier of any one of 17-20 further includes the nucleotide sequence for site-specific integration.
22. 21 carrier, wherein the nucleotides sequence for site-specific integration is classified as and people AAVS1 sites
Homologous nucleotide sequence.
23. the carrier of any one of 17-22, wherein the drug screening gene is antibiotics resistance gene.
24. the carrier of any one of 17-23, wherein the reporter is encoding green fluorescent protein or red fluorescence
The gene of albumen.
25. the carrier of any one of 17-24, wherein all elements in the carrier are all related by the AD is conducive to
Gene expression is ranked sequentially.
26. 25 carrier, wherein all elements in the carrier are all cis arrangement.
27. a kind of genetic constructs, it includes following nucleotide sequences：First promoter；It is controlled by first promoter
Drug screening gene；Second promoter；The AD related genes being connected with the reporter controlled by second promoter；
With the sequence homologous with people AAVS1 sites, wherein said elements are all cis arrangement.
28. 27 genetic constructs, wherein first promoter is PGK-1 or CAG promoters；The drug sieve
It is antibiotics resistance gene to select gene；Second promoter is PGK-1 or CAG promoters；The AD related genes are coding
The gene of BACE1 and the reporter are encoding green fluorescent protein or the gene of red fluorescent protein.
29. 27 genetic constructs, wherein first promoter is PGK-1 or CAG promoters；The drug sieve
It is antibiotics resistance gene to select gene；Second promoter is PGK-1 or CAG promoters；The AD related genes are coding
The gene of PS1dE9 and the gene that the reporter is coding GFP.
30. 28 or 29 genetic constructs, wherein antibiotics resistance gene are puromycin, neomycin, kanamycins
Or genetic resistance genes.
31. the hiPSC converted by the expression vector of any one of item 16-26 or the genetic constructs of any one of item 27-30
Cell line.
32. 31 hiPSC cell lines are used to generate the purposes of AD cell models.
33. the hiPSC of the genetic modification as AD cell models, in people AAVS1 integrations BACE1 genes and
Composition is overexpressed the BACE1 genes of the integration.
34. the hiPSC of 33 genetic modification, with it is no integrate the BACE1 genes wait genes hiPSC compared with,
Show that beta-secretase and/or A β -42 peptides increase.
35. the hiPSC of the genetic modification as AD cell models, in the people AAVS1 integrations PS1dE9 genes
And composition is overexpressed the PS1dE9 genes of the integration.
36. the hiPSC of 35 genetic modification, with it is no integrate the PS1dE9 genes wait genes hiPSC phases
Than showing that beta-secretase and/or A β -42 peptides increase.
37. the hiPSC of any one of 33-36 genetic modification is used for the purposes of high flux screening AD medicines.
38. a kind of method for high flux screening AD therapeutic agents, including
I) expression vector or the hereditary structure of any one of lead in item 27-30 to any one of hiPSC lead in item 16-26 are passed through
Body is built, the cell model of Alzheimer disease (AD) is prepared by hiPSC,
Ii) with above-mentioned cell model culture candidate compound two days to two weeks,
Iii) before and after the candidate compound is added in, measurement beta-secretase level, A β -42 concentration and A β 42/
A β -40 compare；
Wherein, one or more measured values selected from beta-secretase level, A β -42 concentration and A β 42/A β -40 ratios are reduced,
Represent potential treatment agent of the candidate compound for treatment AD.
39. 38 method, the hiPSC comes from people's donor, is converted into vitro by conventional reprogramming method
hiPSC。
40. 39 method, wherein the hiPSC passes through conventional reprogramming from the normal human donor for not suffering from AD
Method is converted into hiPSC in vitro.
41. the high-throughput screening method of any one of 38-40 is used to screen early stage AD drug.
42. a kind of drug screening method for screening beta-secretase or A β -42 inhibitor, including
I) BACE1 genes or PS1dE9 genetic modification hiPSC cell lines are overexpressed by composition,
Ii the hiPSC cell lines) are divided into functional nerve member again,
Iii) cultivated in the presence of candidate drug compounds functional nerve member and
Iv beta-secretase can be reduced by) measuring horizontal beta-secretase, A β 42/A β -40 ratios, and/or A β -42 concentration and screening
Horizontal, A β 42/A β -40 than, and/or A β -42 concentration compound.
43. 42 method, it is included in the presence of candidate drug compounds and cultivates the functional nerve member up to two days
To two weeks.
44. 42 or 43 method, the hiPSC comes from people's donor, is converted in vitro by conventional reprogramming method
For hiPSC.
45. 44 method, wherein the hiPSC passes through conventional reprogramming from the normal human donor for not suffering from AD
Method is converted into hiPSC in vitro.
46. the method for any one of 42-45, wherein by the expression vector to any one of hiPSC lead in item 16-26 or
The genetic constructs of any one of lead in item 27-30 prepare the hiPSC.
Description of the drawings
From being described below and refer to the attached drawing, the these and other aspects and advantage of embodiment of the present invention will become aobvious
And be clear to and be easier to understand, wherein：
The generation of Fig. 1 parent hiPSC cell lines (UCIS3007) and the example of phenotypic evaluation：Separation is from the non-trouble of health
The urine cell of sick donor and by using the standard reprogramming method of 4 kinds of transcription factors (Oct4, Sox2, Klf4 and cMyc)
It is translated into hiPSC cells.A. the process of reprogramming urine cell shows morphological change and alkaline phosphatase (AP) dyeing；
B. the biomarker (Nanog, Tra-1-60, Tra-1-81, SSEA3 and SSEA4) of immunostaining pluripotent stem cell；C-
E. other versatilities test (transgene silencing, the expression of endogenous versatility gene, the FACS analyses of surface marker,
Promoter demethylation (demythelation), karyotyping, embryoid is formed and the expression of germinal layer mark)；With F. monsters
Knurl is formed, and shows three germinal layers.
Neural stem cell and neuronal cell (NSC, composite nerve member, DOPA of Fig. 2 from parent's hiPSC cell lines
Aminergic neuron and motor neuron).
Fig. 3 are used to generate the donor vehicle of the hiPSC cell lines with AD related gene ectopic expressions in AAVS1 sites
Figure.Being related to the nucleotide sequence of targeted integration includes the sequence homologous with people AAVS1 sites (HA-L and HA-R), PGK-1 startups
Son, drug selection marker gene, CAG promoters, AD related genes and reporter, it is all to be arranged by cis fashion.It is different
AD related genes can easily be substituted by using the limitation enzymic digestion of Xho-I and BglII.A. skeleton carrier CIB-
PCBEB；B. it is used for the donor vehicle of targeted integration BACE1 genes；It is carried with C. for the donor of targeted integration PS1dE9 genes
Body.
Fig. 4 targeted integration BACE1 genes enter AAVS1 sites.AAVS1 sites are located at the outer aobvious of PPP1R12C locus
Son 1 and exon 2 include subregion.F1, F2, F3, R1, R2 and R3 are for being verified the primer of connection PCR insertions.
A. targeted integration strategy；B. single cell clone (upper plate is screened by connecting PCR：5 ' ends are carried out using F1+R1 primers to connect
PCR, clone 21 is the IPSN0041-21 with Insert Fragment, and clone 24 is the negative clone of no Insert Fragment；Lower plate：3′
End connects PCR)；5 ' ends of Insert Fragment and 3 ' end sequences in C.IPSN0041-21.
PS1dE9 gene targets are integrated into AAVS1 sites by Fig. 5.AAVS1 sites are located at the outer of PPP1R12C locus
Aobvious son 1 and exon 2 are included in subregion.F1, F2, F3, R1, R2 and R3 are for by connecting drawing for PCR verification insertions
Object.A. targeted integration strategy；B. single cell clone (upper plate is screened by connecting PCR：5 ' ends are carried out using F1+R1 primers to connect
Meet PCR.74 be the clone with the Insert Fragment for further characterization of selection, and 67 be not inserted into segment negative gram
It is grand；Lower plate：3 ' ends connect PCR)；C. 5 ' ends of Insert Fragment and the sequence of 3 ' ends in clone 74；D.iPSC
(UCIS3007-74) the PS1dE9 gene overexpressions in.
Fig. 6 are prepared the preparation method of a large amount of neuronal cells by HiPSC, and display neural stem cell (NSC) is as intermediate
Stage is the committed step for shortening continuous mode and reducing variation.Neural stem cell NSC specific biomarkers sox-1
(red, to dye core) and nestin (green, staining cell matter) carry out immunostaining.Functional nerve member neuron-specific
Property biomarker TujI (red) and Dapi (blueness) carry out immunostaining.
Compared with parental cell system IPSN0041, BACE1 gene overexpressions in IPSN0041-21 are shown Fig. 7 simultaneously
During IPSN0041-21 is compared with parental cell system IPSN0041, BACE1 genes have apparent on RNA and protein level
Higher expression.The mRNA tables between IPSN0041-21 and IPSN0041 in A.hiPSC and neuron from hiPSC
It reaches；B. the BACE1 protein expressions in neural stem cell and neuron from hiPSC cell lines.
Fig. 8 are compared with IPSN0041, the overexpression of A β -42 peptides in IPSN0041-21.A. the neuron from hiPSC
In A β-42 concentration, show the neuron from IPSN0041-21 have it is more higher A β than the neuron from IPSN0041-
42 concentration；The time-histories research that A β -42 are expressed between B.IPSN0041-21 and IPSN0041, shows different expression patterns.
Fig. 9 use the drug screening embodiment of IPSN0041-21, the two kinds of small molecule chemical combination do not reported before display
Object, B3 and B16 reduce the generation of A β -40 and A β -42 peptides in the neuron from hiPSC, but do not influence A β -42/40 ratios.Make
By the use of known beta-secretase inhibitor (10uM) as positive control.Data are repeated from three biology.A.B3 and B16 subtract
The generation of few A β -40；B.B3 and B16 reduces the generation of A β -42；With C.A β -42 compared with the ratio of A β -40
Specific embodiment
Unless otherwise defined, all technical and scientific terms used in this application have and those of ordinary skill in the art
Normally understood identical meaning.For example, for application provide biological field in term, researcher with particular reference to
Sambrook et al., Molecular Cloning:A Laboratory Manual, the second edition, Cold Spring Harbor
Press,Plainsview,New York(1989)；With Ausubel et al., Current Protocols in Molecular
Biology(Supplement 47),John Wiley& Sons,New York(1999).These terms should not be interpreted as having
The smaller scope understood than those of ordinary skill in the art.
Term " comprising " is used in the specification and claims, not exclude other elements or step.It is unless another
It clearly states, when referring to singular noun using indefinite article or definite article "one" or " one kind ", " this ", " this ",
It includes the plural number of the noun.
" AD related genes " represents the relevant any gene of Ahl tribulus sea silent sickness (AD) morbidity.Although for A Erci
The cause of disease understanding that sea is write from memory sick is very few, but thinks that about 70% risk and many genes being usually directed to have genetic affinity, the base
Because of such as mutator of encoding amyloid precursor albumen (APP) and presenilin (PS) 1 and 2.App gene mutation causes different
Normal APP albumen, the exception APP albumen are preferentially cut by beta-secretase, generate more A β peptides；And PS gene mutations cause
Preferential to generate 42 peptides of A β, 42 peptides of A β are the ingredient of amyloid patch.Although it is not sent out in FAD or SAD patients
The gene for now encoding beta-secretase is undergone mutation, but clearly finds that raised BACE1 expression causes the expression of A β peptide to raise,
So as to increase the level of A β -42 peptides in AD patient.These genes, it is closely related with AD morbidities together with finding or will be seen that
Other genes, the AD related genes being referred to as in the present invention.
Mutant amyloid precursor albumen (APP) or 1 and 2 gene representation of presenilin (PS) in the application cause AD to fall ill
Mutation APP or PS1 or PS2 gene.The mutation of these genes can be naturally occurring in AD patient or heredity production in vitro
It is raw.Described in the present invention during term " BACE1 genes ", not only include naturally occurring gene, but also including the change of its function
Body.Functional variety can be slightly different with naturally occurring gene in amino acid sequence, for example, with naturally occurring BACE1 eggs
White amino acid sequence has at least 80%, preferably at least 90%, more preferably at least 95%, 96%, 97%, 98%, 99%
Homology, but they still have the function of it is identical with natural B ACE1 albumen.
" constructive expression " refers to the gene table of the consecutive transcription compared with only according to the facultative gene transcribed
It reaches." overexpression " refers to excessively high or excessive gene expression dose, generates apparent gene-correlation phenotype.With regard to the present invention
Speech is integrated into the AD related genes in hiPSC and is overexpressed in hiPSC for composition, and in amyloid precusor protein
Processing procedure in cause AD phenotypes occur.
" the AD phenotypes " of the present invention is related with the phenotype in amyloid precusor protein processing procedure, and the phenotype passes through
The AD related genes composition integrated AD related genes and integrated is overexpressed induction and generates, and the AD phenotypes include but unlimited
In the horizontal increase of beta-secretase, amyloid beta peptide increases, A β -42 concentration increases and/or A β 42/A β -40 increase.
In the present invention, the AD related genes are integrated into hiPSC is overexpressed the AD related genes for composition,
To generate AD phenotypes.It is preferred that by site-specific fashion, most preferably by using the sequence, that is, people homologous with safe port site
AAVS1 is integrated in site." homologous " of gene is needed to realize homologous recombination, so as to by exogenous origin gene integrator to target site.
Based on common knowledge, skilled person will know how designs and the nucleic acid of the nucleic acid sequence homologous on targeted integration site
Sequence, to realize the purpose of homologous recombination.Therefore, the present invention includes but not limited to embodiment institute when mentioning homologous sequence
The homologous sequence used, while it can be that realization is whole in people AAVS1 sites by routine techniques to further include those skilled in the art
Any homologous sequence for closing and designing.
Carrier
The AD cell models of the present invention can be generated using genetic recombinant methods, and AD related genes are imported into and are come from
In the hiPSC of non-diseased person.AD cell models are generated for restructuring, APP the or PS eggs of AD GAP-associated protein GAPs such as mutation will be encoded
White or BACE1 enzymes nucleic acid is separated and is inserted into replicable vector, for further cloning (DNA amplification) or table
It reaches.The DNA of encoding said proteins can be easily separated and be sequenced using the conventional method of this field.
For the purposes of the present invention, many carriers can be used for further transformation.Component in carrier generally includes but not
It is limited to one or more of following：Replication orgin, one or more marker gene, one or more reporter genes, enhancer
Element, promoter and transcription terminator.
Replication orgin
Both expression and cloning vector contain enables the nucleotide sequence that carrier replicates in selected host cell.
In general, in cloning vector, which is the sequence that carrier is enable to be replicated independently of host chromosome DNA,
And including replication orgin or autonomously replicating sequence.This sequence is well-known for various bacteriums, yeast and virus.
Replication orgin from pBR322 plasmid is suitable for most of gramnegative bacteriums, and 2 μ plasmid origins are suitable for yeast, various
The cloning vector that viral origins (SV40, polyoma, adenovirus, VSV or BPV) can be used in mammalian cell.In general, lactation
Animal expression vector need not replicate ingredient starting point (can be usually using SV40 starting points, simply because it contains early promoter).
Select gene element
Expression and cloning vector can contain selection gene, also referred to as selected marker.Under typical selection gene code
State protein：Its (a) assigns the resistance for antibiotic or other toxin, such as ampicillin, neomycin, methotrexate (MTX)
Or tetracycline, uracil, kanamycins and Geneticin；(b) extra-nutrition deficiency defect；Or (c) is provided from compound criteria
The unavailable critical nutrients of base, such as the gene of the D-alanine racemase of coding bacillus.
One example of selection scheme is using drug to prevent the growth of host cell.With heterologous gene successful conversion
Those cells generate the albumen for assigning drug resistance, so as to survive in selection scheme.The example of this dominant selection uses drug
Neomycin, urea, kanamycins and Geneticin and hygromycin.
Another example for the suitable selected marker of mammalian cell is can to identify to have the ability to absorb to compile
The cell of the antibody of code nucleic acid, such as DHFR, glutamine synthelase (GS), thymidine kinase, metallothionein-I and-II, it is excellent
Select primate metallothionein's gene, 20 deaminase of adenosine, ornithine decarboxylase etc..
For example, by cultivating transformant in the culture medium of the competitive antagonist containing methotrexate (MTX) (Mtx), DHFR
To identify the cell with DHFR genetic transformation.Under these conditions, DHFR genes carry out together with any other cotransformation nucleic acid
Amplification.
Alternatively, by cultivating transformant in the culture medium containing l-methionine sulphoxide imine (Msx), GS inhibitor
To identify the cell with GS genetic transformation.Under these conditions, GS genes are expanded together with any other cotransformation nucleic acid
Increase.GS selections/amplification system can be used in combination with above-mentioned DHFR selections/amplification system.
Reporter ingredient
Reporter (usual abbreviation reporter molecule) is the adjusting that researcher invests organism another gene of interest
Gene in sequence.Some genes are selected as reporter molecule, because these genes assign the feature for expressing their organism
Be easy to identify and measure or because these genes be selected marker.Reporter be commonly used as indicating a certain gene whether by
Cell or biocenose receive or expression.
In order to which reporter is introduced organism, reporter and target gene are placed in identical DNA and built by scientist
To be inserted into cell or organism in body.The reporter of common induction visual identification feature is usually directed to fluorescence and shines
Albumen.Example includes the gene of coding jellyfish green fluorescent albumen (GFP) and the red fluorescent protein from gene dsRed,
The jellyfish green fluorescent protein gene can cause the cell green light under blue light for expressing this gene.
Start subconstiuent
Expression and cloning vector usually contain promoter, and the promoter is identified by host cell and can grasped
It is connected to the nucleic acid of coding destination protein with making.
Promoter sequence is known for eucaryote.All there are one be rich in AT for almost all of eukaryotic gene
Region, the region is approximately at the base of starting transcription site upstream.70 from the upstream of many genes transcripting start point
It is CNCAAT regions to another sequence obtained by 80 bases, wherein N can be any nucleotide.In most of eucaryon bases
The 3' ends of cause are AATAAA sequences, can be the signals for the 3' ends that polyA tails are added to coded sequence.It is all this
A little sequences are all properly inserted into carrier for expression of eukaryon.
The AD pathogenic proteins matter transcription of carrier in hiPSC can be controlled for example by following promoter：From virus
The promoter that genome obtains, virus such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), the cow teats
Tumor virus, avian sarcomata virus, cytomegalovirus, retrovirus, hepatitis type B virus, simian virus 40 (SV40)；Or from different
The promoter that source mammalian promoter obtains, such as actin promoter or immunological defence；It is obtained from heat-shock promoters
The promoter obtained, condition is that this promoter is compatible with host cell systems.
The early and late promoter of SV40 viruses can be restricted as the SV40 comprising SV40 virus origin of replication
Section easily obtains.The instant early promoter of human cytomegalovirus is easily obtained as HindIII E restriction fragments.It is beautiful
The system for bovine papilloma virus being used disclosed in state's patent 4,419,446 as carrier DNA being expressed in mammalian hosts.
The improvement of the system described in United States Patent (USP) 4,601,978.Referring also to Reyes et al., Nature 297:598-601
(1982), on people's p- interferon cDNA in the mouse cell under the control of 5 promoter of thymidine kinase of herpes simplex virus
Expression.Alternatively, the long terminal of Rous sarcoma virus repeats to may be used as promoter.
CAG promoters are powerful synthetic promoters, and the gene being frequently used in driving mammalian expression vector is high
Horizontal expression37-38.It is from following sequence construct：
(C) cytomegalovirus (CMV) early stage enhancer element,
(A) First Intron of promoter, First Exon and avian beta-actin gene,
(G) acceptor splicing site of rabbit beta-globin gene.
The synthin of gained is used for pCAGGS expression vectors.
Although entire construct is commonly referred to as " CAG promoters ", it is not proper promoter, because it
A part comprising transcription sequence and introne) and enhancer element.In addition to the instant early stage enhancers of CMV, chicken β fleshes move
The introne of protein gene also contains enhancer element highly conserved in vertebrate.The 3' parts of promoter have height
G/C content, therefore be not suitable for PCR amplification.
PGK-1 gene codes are run one's home enzyme, glycerol 3-phosphate acid kinase, and are wide expressions.The gene is located at lactation
On the X chromosome of animal, and except its with it is most on the inactive X chromosome of female somatic cells or male sex-cell
The other genes of number outside silence, are always expressed together.- 1 promoters of Pgk (PGK) are located at the region rich in nucleotide G and C
It is interior.The promoter can effectively drive the high level expression of the reporters such as Escherichia coli lacZ and neo.Transcription initiation position
The 120bp of point upstream is as core promoter.Its upstream is the region of 320bp, is increased in a manner of being orientated with position independence
The transcription of strong core promoter.The region of the 320bp does not enhance the transcription of SV40 early stage areas core promoter.Nucleoprotein is with being somebody's turn to do
320bp segments combine, but gel mobility shift assay method can be used to prove the restricted area combined, so as to show to enhance
The activity of son can be mediated by the factor that multiple sites combine, and each site has low-affinity39。
Enhancer element ingredient
By the way that enhancer sequence is inserted into carrier, the DNA transcriptions of the coding destination protein in higher eucaryote often increase
Add.Now know many enhancers from mammalian genes (globin, elastoser, albumin, alpha-fetoprotein and insulin)
Sequence.However, the enhancer from eukaryotic cell virus would generally be used.Example is included on rear side of replication orgin (100-270bp)
SV40 enhancers, the sub- enhancer of cytomegalovirus early promoter, the polyoma enhancer on rear side of compound starting point and adenovirus enhancing
Son.Referring also to Yaniv, Nature 297:Reinforcing elements of the 17-18 (1982) on activation eukaryotic promoter.It can be anti-
5' or 3' of body coded sequence but the 5' sites of promoter are preferably placed at, by enhancer montage into carrier.
Transcription termination component
It will also contain for the expression vector in eukaryotic host cell such as people's cell and terminate needed for transcription and stable mRNA
Sequence.This sequence usually can be from the 5' of eucaryon or viral DNA or cDNA and the non-translational region of 3' obtains once in a while.These areas
Domain is included in the nucleotide segment that polyadenylated fragments are transcribed into the untranslated part of the mRNA of coding destination protein.
In one embodiment, carrier of the invention is designed for Ahl tribulus sea silent sickness (AD) related gene is had
The gene efficient rate of pass targeted integration into people's pluripotent stem cell (hiPSC).Therefore, carrier is also designed to include and be used for
The sequence of site-specific integration.
It is that a kind of genome orients editing technique to be inserted into gene in known location by the enzyme with targets identification ability,
Researcher can be made to delete, be inserted into high Efficiency and accuracy for the technology or any gene or DNA of modifier group level
Segment40-42.What this technology was additionally included in for example disclosed in US8697359, US8771945, US8795965 nearest makes extensively
CRISPRcas9 gene editing technologies.In some embodiments of the present invention, AD related genes are passed through
CRISPRcas9 gene editing technology specific integrations are to safe port site, i.e. AAVS1 sites.
AAVS1 sites are the natural A AV integration sites on human chromosome 19.The region (AAVS1), which has to become, to be turned
The feature of the ideal targets of gene.
In the specific embodiment of the present invention, carrier includes the drug selection marker controlled by the first promoter
Gene, AD related genes being connected with the reporter controlled by the second promoter and same with the sequence in people AAVS1 sites
The sequence in source.
In the specific embodiment of the present invention, donor vehicle main chain includes whole for targeting by cis arrangement
The left arm (HA-L) of conjunction, PGK-1 promoters, purine radicals because, CAG promoters, GFP reporters and the right side for targeted integration
Arm (HA-RL) (Fig. 4 A).
As used herein, following sequence is included for the left arm (HA-L) of targeted integration：
TGCTTTCTCTGACCAGCATTCTCTCCCCTGGGCCTGTGCCGCTTTCTGTCTGCAGCTTGTG
GCCTGGGTCACCTCTACGGCTGGCCCAGATCCTTCCCTGCCGCCTCCTTCAGGTTCCGTC
TTCCTCCACTCCCTCTTCCCCTTGCTCTCTGCTGTGTTGCTGCCCAAGGATGCTCTTTCCG
GAGCACTTCCTTCTCGGCGCTGCACCACGTGATGTCCTCTGAGCGGATCCTCCCCGTGTC
TGGGTCCTCTCCGGGCATCTCTCCTCCCTCACCCAACCCCATGCCGTCTTCACTCGCTGGG
TTCCCTTTTCCTTCTCCTTCTGGGGCCTGTGCCATCTCTCGTTTCTTAGGATGGCCTTCTCC
GACGGATGTCTCCCTTGCGTCCCGCCTCCCCTTCTTGTAGGCCTGCATCATCACCGTTTTT
CTGGACAACCCCAAAGTACCCCGTCTCCCTGGCTTTAGCCACCTCTCCATCCTCTTGCTTT
CTTTGCCTGGACACCCCGTTCTCCTGTGGATTCGGGTCACCTCTCACTCCTTTCATTTGGG
CAGCTCCCCTACCCCCCTTACCTCTCTAGTCTGTGCTAGCTCTTCCAGCCCCCTGTCATGG
CATCTTCCAGGGGTCCGAGAGCTCAGCTAGTCTTCTTCCTCCAACCCGGGCCCCTATGTC
CACTTCAGGACAGCATGTTTGCTGCCTCCAGGGATCCTGTGTCCCCGAGCTGGGACCAC
CTTATATTCCCAGGGCCGGTTAATGTGGCTCTGGTTCTGGGTACTTTTATCTGTCCCCTCC
ACCCCACAGTGGGGC。
PGK- puromycins box includes following sequence：
ATAACTTCGTATAATGTATGCTATACGAAGTTATTACCGGGTAGGGGAGGCGCTTTTCCCA
AGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTG
GCCTCTGGCCTCGCACACATTCCACATCCCCCGGTAGGCGCCAACCGGCTCCGTTCTTTG
GTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGC
AGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAG
ATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGC
AGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCG
GGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCG
GCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGG
GCCTTTCGGAATTCATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGAC
GTCCCCCGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCA
CACCGTCGACCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCA
CGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGG
CGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCC
CGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCT
CCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCG
CCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCG
GCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTT
CTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGC
ACCTGGTGCATGACCCGCAAGCCCGGTGCCTGATAGAACTTGTTTATTGCAGCTTATAAT
GGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATT
CTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGATAACTTCGTATAATGT
ATGCTATACGAAGTTAT。
CAG promoters include following sequence：
ATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATA
TGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACC
CCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCA
TTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTA
TCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTAT
GCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCG
CTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTC
CCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGG
GGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGG
CGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTT
TTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGG
GAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCG
CCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCC
TCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGA
AAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTG
CGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTG
TGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAG
CGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACAAAGGCTG
CGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGC
TGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCG
GGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCA
GGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAG
GGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGTCGCAGCCAT
TGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGTG
GAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGG
TGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGC
CGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGGCGGCTGCCTTCGGG
GGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCT
CTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATT
GTGCTGTCTCATCATTTTGGCAAAGAATTCCTCGACCTCGAG。
GFP reporters include following sequence：
AGATCTGGCAGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAG
AATCCCGGCCCTAGGTTCGAAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGG
TGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGG
CGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACC
GGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTGACCTACGGCGTGCAGTG
CTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGA
AGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGC
GCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCG
ACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCA
CAAGGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGACCC
GCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCC
CATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCC
TGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGC
CGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTGA。
Following sequence is included for the right arm (HA-RL) of targeted integration：
TACTAGGGACAGGATTGGTGACAGAAAAGCCCCATCCTTAGGCCTCCTCCTTCCTAGTCT
CCTGATATTGGGTCTAACCCCCACCTCCTGTTAGGCAGATTCCTTATCTGGTGACACACCC
CCATTTCCTGGAGCCATCTCTCTCCTTGCCAGAACCTCTAAGGTTTGCTTACGATGGAGCC
AGAGAGGATCCTGGGAGGGAGAGCTTGGCAGGGGGTGGGAGGGAAGGGGGGGATG
CGTGACCTGCCCGGTTCTCAGTGGCCACCCTGCGCTACCCTCTCCCAGAACCTGAGCTGC
TCTGACGCGGCTGTCTGGTGCGTTTCACTGATCCTGGTGCTGCAGCTTCCTTACACTTCC
CAAGAGGAGAAGCAGTTTGGAAAAACAAAATCAGAATAAGTTGGTCCTGAGTTCTAACT
TTGGCTCTTCACCTTTCTAGTCCCCAATTTATATTGTTCCTCCGTGCGTCAGTTTTACCTGT
GAGATAAGGCCAGTAGCCAGCCCCGTCCTGGCAGGGCTGTGGTGAGGAGGGGGGTGT
CCGTGTGGAAAACTCCCTTTGTGAGAATGGTGCGTCCTAGGTGTTCACCAGGTCGTGGC
CGCCTCTACTCCCTTTCTCTTTCTCCATCCTTCTTTCCTTAAAGAGTCCCCAGTGCTATCTG
GGACATATTCCTCCGCCCAGAGCAGGGTCCCGCTTCCCTAAGGCCCTGCTCTGGGCTTCT
GGGTTTGAGTCCTTGGCAAGCCCAGGAGAGGCGCTCAGGCTTCCCTGTCCCCCTTCCTC
GTCCACCATCTCATGCCCCTGGCTCTCCTGCCCCTTCCCTACAGGGGTTCCTGGCTCTGCT CT。
In another specific embodiment, donor vehicle is included in BgIII and XhoI sites insertion carrier framework
BACE1 genes (Fig. 4 B)；The cDNA sequence of BACE1 genes is disclosed in Genbank, is locus NM_138973.3 (Homo
Sapiens beta-secretases 1, transcriptional variants d, mRNA).As used herein, " BACE1 " represents the cross-film by BACE1 gene codes
Protease.BACE1 is catalyzed the first step that amyloid beta is formed by amyloid precusor protein.Amyloid beta peptide is amyloid
The main component of albumen beta plaque is accumulated in the brain of mankind's patients with Alzheimer disease.
The DNA sequence dna of BACE1 genes is described as in donor vehicle：
ATGGCCCAAGCCCTGCCCTGGCTCCTGCTGTGGATGGGCGCGGGAGTGCTGCCTGCCCA
CGGCACCCAGCACGGCATCCGGCTGCCCCTGCGCAGCGGCCTGGGGGGCGCCCCCCTG
GGGCTGCGGCTGCCCCGGGAGACCGACGAAGAGCCCGAGGAGCCCGGCCGGAGGGG
CAGCTTTGTGGAGATGGTGGACAACCTGAGGGGCAAGTCGGGGCAGGGCTACTACGTG
GAGATGACCGTGGGCAGCCCCCCGCAGACGCTCAACATCCTGGTGGATACAGGCAGCA
GTAACTTTGCAGTGGGTGCTGCCCCCCACCCCTTCCTGCATCGCTACTACCAGAGGCAGC
TGTCCAGCACATACCGGGACCTCCGGAAGGGTGTGTATGTGCCCTACACCCAGGGCAAG
TGGGAAGGGGAGCTGGGCACCGACCTGCTTTGTGGTGCTGGCTTCCCCCTCAACCAGT
CTGAAGTGCTGGCCTCTGTCGGAGGGAGCATGATCATTGGAGGTATCGACCACTCGCTG
TACACAGGCAGTCTCTGGTATACACCCATCCGGCGGGAGTGGTATTATGAGGTGATCATT
GTGCGGGTGGAGATCAATGGACAGGATCTGAAAATGGACTGCAAGGAGTACAACTATG
ACAAGAGCATTGTGGACAGTGGCACCACCAACCTTCGTTTGCCCAAGAAAGTGTTTGAA
GCTGCAGTCAAATCCATCAAGGCAGCCTCCTCCACGGAGAAGTTCCCTGATGGTTTCTG
GCTAGGAGAGCAGCTGGTGTGCTGGCAAGCAGGCACCACCCCTTGGAACATTTTCCCA
GTCATCTCACTCTACCTAATGGGTGAGGTTACCAACCAGTCCTTCCGCATCACCATCCTTC
CGCAGCAATACCTGCGGCCAGTGGAAGATGTGGCCACGTCCCAAGACGACTGTTACAAG
TTTGCCATCTCACAGTCATCCACGGGCACTGTTATGGGAGCTGTTATCATGGAGGGCTTC
TACGTTGTCTTTGATCGGGCCCGAAAACGAATTGGCTTTGCTGTCAGCGCTTGCCATGTG
CACGATGAGTTCAGGACGGCAGCGGTGGAAGGCCCTTTTGTCACCTTGGACATGGAAG
ACTGTGGCTACAACATTCCACAGACAGATGAGTCAACCCTCATGACCATAGCCTATGTCAT
GGCTGCCATCTGCGCCCTCTTCATGCTGCCACTCTGCCTCATGGTGTGTCAGTGGCGCTG
CCTCCGCTGCCTGCGCCAGCAGCATGATGACTTTGCTGATGACATCTCCCTGCTGA。
It is described as with the protein sequence of BACE1： MAQALPWLLLWMGAGVLPAHGTQHGIRLPLRSGLGGAPLGLR
LPRETDEEPEEPGRRGSF VEMVDNLRGKSGQGYYVEMTVGSPPQTLNILVDTGSSNFAVGAAPHPFLHRYYQRQLS
ST YRDLRKGVYVPYTQGKWEGELGTDLLCGAGFPLNQSEVLASVGGSMIIGGIDHSLYTGSLW
YTPIRREWYYEVIIVRVEINGQDLKMDCKEYNYDKSIVDSGTTNLRLPKKVFEAAVKSIKAAS
STEKFPDGFWLGEQLVCWQAGTTPWNIFPVISLYLMGEVTNQSFRITILPQQYLRPVEDVA
TSQDDCYKFAISQSSTGTVMGAVIMEGFYVVFDRARKRIGFAVSACHVHDEFRTAVEGPFV
TLDMEDCGYNIPQTDESTLMTIAYVMAAICALFMLPLCLMVCQWRCLRCLRQQHDDFAD DISLLK。
In another specific embodiment, donor vehicle includes BgIII the and XhoI sites in insertion carrier framework
PS1dE9 genes (Fig. 5 B).PS1dE9 is the mutant of the wherein PS1 genes that PS1 gene extrons 9 lack.As used herein,
Term " PS1 " represents the protein by 1 gene code of presenilin.The cDNA sequence of PS1 genes is disclosed as gene in Genbank
Seat NM_000021.3 (Homo sapiens, presenilin 1, transcriptional variants 1, mRNA).Presenilin 1 is four in presenilin compound
One of kind core protein, the regulatory protein hydrolysis event of some protein in mediated cell, including gamma secretase.Term
" PS1dE9 " represents the mutator of PS1, and abnormal gamma-secretase is caused to crack, is conducive to the generation of A β -42 peptides, from
And the early stage of Alzheimer's disease is caused to be fallen ill.
The DNA sequence dna of PS1dE9 is (highlighting as deletion sequence) as described below in donor vehicle：
ATGACAGAGTTACCTGCACCGTTGTCCTACTTCCAGAATGCACAGATGTCTGAGGACAAC
CACCTGAGCAATACTGTACGTAGCCAGAATGACAATAGAGAACGGCAGGAGCACAACGA
CAGACGGAGCCTTGGCCACCCTGAGCCATTATCTAATGGACGACCCCAGGGTAACTCCC
GGCAGGTGGTGGAGCAAGATGAGGAAGAAGATGAGGAGCTGACATTGAAATATGGCG
CCAAGCATGTGATCATGCTCTTTGTCCCTGTGACTCTCTGCATGGTGGTGGTCGTGGCTAC
CATTAAGTCAGTCAGCTTTTATACCCGGAAGGATGGGCAGCTAATCTATACCCCATTCACA
GAAGATACCGAGACTGTGGGCCAGAGAGCCCTGCACTCAATTCTGAATGCTGCCATCAT
GATCAGTGTCATTGTTGTCATGACTATCCTCCTGGTGGTTCTGTATAAATACAGGTGCTATA
AGGTCATCCATGCCTGGCTTATTATATCATCTCTATTGTTGCTGTTCTTTTTTTCATTCATTTA
CTTGGGGGAAGTGTTTAAAACCTATAACGTTGCTGTGGACTACATTACTGTTGCACTCCT
GATCTGGAATTTTGGTGTGGTGGGAATGATTTCCATTCACTGGAAAGGTCCACTTCGACT
CCAGCAGGCATATCTCATTATGATTAGTGCCCTCATGGCCCTGGTGTTTATCAAGTACCTCC
CTGAATGGACTGCGTGGCTCATCTTGGCTGTGATTTCAGTATATGATTTAGTGGCTGTTTT
GTGTCCGAAAGGTCCACTTCGTATGCTGGTTGAAACAGCTCAGGAGAGAAATGAAACGC
TTTTTCCAGCTCTCATTTACTCCT CAAGTATAATGCAGAAAGCACAG
AAAGGGAGTCACAAGACACTGTTGCAGAGAATGATGATGGCGGGTTCAGTGAGGAATG
GGAAGCCCAGAGGGACAGTCATCTAGGGCCTCATCGCTCTACACCTGAGTCACGAGCTG
CTGTCCAGGAACTTTCCAGCAGTATCCTCGCTGGTGAAGACCCAGAGGAAAGGGGAGT
AAAACTTGGATTGGGAGATTTCATTTTCTACAGTGTTCTGGTTGGTAAAGCCTCAGCAAC
AGCCAGTGGAGACTGGAACACAACCATAGCCTGTTTCGTAGCCATATTAATTGGTTTGTG
CCTTACATTATTACTCCTTGCCATTTTCAAGAAAGCATTGCCAGCTCTTCCAATCTCCATCA
CCTTTGGGCTTGTTTTCTACTTTGCCACAGATTATCTTGTACAGCCTTTTATGGACCAATTA
GCATTCCATCAATTTTATATCTAG。
(highlighted is since missing extron 9 causes erroneous translation as described below with the protein sequence of PS1dE9
Part)：
MTELPAPLSYFQNAQMSEDNHLSNTVRSQNDNRERQEHNDRRSLGHPEPLSNGRPQGN
SRQVVEQDEEEDEELTLKYGAKHVIMLFVPVTLCMVVVVATIKSVSFYTRKDGQLIYTPFTED
TETVGQRALHSILNAAIMISVIVVMTILLVVLYKYRCYKVIHAWLIISSLLLLFFFSFIYLGEVFKT
YNVAVDYITVALLIWNFGVVGMISIHWKGPLRLQQAYLIMISALMALVFIKYLPEWTAWLIL
AVISVYDLVAVLCPKGPLRMLVETAQERNETLFPALIYSS
In yet another embodiment of the present invention, donor vehicle of the invention can include any variant of BACE1
And/or any variant of PS1.Variant includes the allele variant (such as polymorphism) of such as Different Individual, optional montage shape
Naturally occurring variant caused by formula etc..Variant preferably substantially with sequence homology according to the present invention, that is, is shown and the present invention
Sequence typically at least about 80%, preferably at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, 99% core
Nucleotide sequence homogeneity.The variant of gene of the present invention be also included under stringent hybridization condition with sequence as defined above (or its mutually
Mend chain) hybridization nucleotide sequence.Typical stringent hybridization condition includes temperature higher than 42 DEG C, and salinity is equal to or less than 200mM.
HiPSC cell lines
In one embodiment, the present invention relates to the method by the present invention is whole in safe port site (AAVS1 sites)
It closes AD related genes and generates hiPSC cell lines.The hiPSC cell lines composition that the present invention creates is overexpressed the gene integrated, and
And display, compared with equivalent gene compares hiPSC cell lines, beta-secretase and/or A β -42 peptides increase.
In a detailed embodiment, hiPSC cell lines prepared by the method for the present invention have at AAVS1
The exogenous nucleic acid sequences that point is integrated.Nucleotide sequence includes PGK-1 promoters, puromycin gene, CAG promoters, BACE1 bases
Cause and GFP genes and the sequence for connecting different fragments.The nucleotide sequence of insertion is as described below, and (part that underscore is shown is
BACE1 genes)：
ATAACTTCGTATAATGTATGCTATACGAAGTTATTACCGGGTAGGGGAGGCGCTTTTCCCA
AGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTG
GCCTCTGGCCTCGCACACATTCCACATCCCCCGGTAGGCGCCAACCGGCTCCGTTCTTTG
GTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGC
AGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAG
ATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGC
AGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCG
GGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCG
GCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGG
GCCTTTCGGAATTCATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGAC
GTCCCCCGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCA
CACCGTCGACCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCA
CGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGG
CGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCC
CGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCT
CCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCG
CCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCG
GCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTT
CTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGC
ACCTGGTGCATGACCCGCAAGCCCGGTGCCTGATAGAACTTGTTTATTGCAGCTTATAAT
GGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATT
CTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGATAACTTCGTATAATGT
ATGCTATACGAAGTTATGCGGCCGCAATCGTCGACCTGCAGGCATGCAAGCTTATTGATTA
TTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTT
CCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCC
ATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGT
CAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGC
CAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGT
ACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACC
ATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCC
CAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGG
GGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCG
GAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCG
AGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCT
GCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCT
CTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCT
GTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTG
AGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTG
TGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCT
GCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCC
GGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGG
GGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACC
CCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCC
GTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGG
GTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCG
GCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGTCGCAGCCATTGCCTTTT
ATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGTGGAGCCGA
AATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCG
CCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCC
TTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGGCGGCTGCCTTCGGGGGGGACG
GGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAAC
CATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGT
CTCATCATTTTGGCAAAGAATTCCTCGACCTCGAGATGGCCCAAGCCCTGCCCTGGCTCCTGCTGTGGATGGGCGCG GGAGTGCTGCCTGCCCACGGCACCCAGCACGGCATCCGGCTGCCCCTGCGCAGCGGCCTGGGGGGCGCCCCCCTGGG GCTGCGGCTGCCCCGGGAGACCGACGAAGAGCCCGAGGAGCCCGGCCGGAGGGGCAGCTTTGTGGAGATGGTGGACA ACCTGAGGGGCAAGTCGGGGCAGGGCTACTACGTGGAGATGACCGTGGGCAGCCCCCCGCAGACGCTCAACATCCTG GTGGATACAGGCAGCAGTAACTTTGCAGTGGGTGCTGCCCCCCACCCCTTCCTGCATCGCTACTACCAGAGGCAGCT GTCCAGCACATACCGGGACCTCCGGAAGGGTGTGTATGTGCCCTACACCCAGGGCAAGTGGGAAGGGGAGCTGGGCA CCGACCTGCTTTGTGGTGCTGGCTTCCCCCTCAACCAGTCTGAAGTGCTGGCCTCTGTCGGAGGGAGCATGATCATT GGAGGTATCGACCACTCGCTGTACACAGGCAGTCTCTGGTATACACCCATCCGGCGGGAGTGGTATTATGAGGTGAT CATTGTGCGGGTGGAGATCAATGGACAGGATCTGAAAATGGACTGCAAGGAGTACAACTATGACAAGAGCATTGTGG ACAGTGGCACCACCAACCTTCGTTTGCCCAAGAAAGTGTTTGAAGCTGCAGTCAAATCCATCAAGGCAGCCTCCTCC ACGGAGAAGTTCCCTGATGGTTTCTGGCTAGGAGAGCAGCTGGTGTGCTGGCAAGCAGGCACCACCCCTTGGAACAT TTTCCCAGTCATCTCACTCTACCTAATGGGTGAGGTTACCAACCAGTCCTTCCGCATCACCATCCTTCCGCAGCAAT ACCTGCGGCCAGTGGAAGATGTGGCCACGTCCCAAGACGACTGTTACAAGTTTGCCATCTCACAGTCATCCACGGGC ACTGTTATGGGAGCTGTTATCATGGAGGGCTTCTACGTTGTCTTTGATCGGGCCCGAAAACGAATTGGCTTTGCTGT CAGCGCTTGCCATGTGCACGATGAGTTCAGGACGGCAGCGGTGGAAGGCCCTTTTGTCACCTTGGACATGGAAGACT GTGGCTACAACATTCCACAGACAGATGAGTCAACCCTCATGACCATAGCCTATGTCATGGCTGCCATCTGCGCCCTC TTCATGCTGCCACTCTGCCTCATGGTGTGTCAGTGGCGCTGCCTCCGCTGCCTGCGCCAGCAGCATGATGACTTTGC TGATGACATCTCCCTGCTGAAGGTCGACAGATCTGGCAGCGGAGAGGGC
AGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGTTCGAAAT
GGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGA
CGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCAC
CTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGC
CCACCCTCGTGACCACCTTGACCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACA
TGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACC
ATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCG
ACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACAT
CCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAGGTCTATATCACCGCCGACA
AGCAGAAGAACGGCATCAAGGTGAACTTCAAGACCCGCCACAACATCGAGGACGGCAG
CGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGC
TGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAA
GCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGG
ACGAGCTGTACAAGTGAGAGCTCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTT
TGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAAT
AAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGG
GTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCGATCG。
The sequence of 5' ends junction between Insert Fragment and host genome is expressed as：
ACCCCACAGTGGGGCAAGCTTGGATCCTC and its complementary series.
The sequence of 3' ends junction between Insert Fragment and host genome is expressed as：
CGATCGGCGGCCGCTACTAGGGACAGGATT and its complementary series.
In a detailed embodiment, hiPSC cell lines prepared by the method for the present invention have at AAVS1
The exogenous nucleic acid sequences that point is integrated.Nucleotide sequence includes PGK-1 promoters, puromycin gene, CAG promoters, mutant
PS1 genes (PS1dE9) and GFP genes and the sequence for connecting different fragments.The nucleotide sequence of insertion (underscore as described below
The part of display is PS1dE9 genes)： ATAACTTCGTATAATGTATGCTATACGAAGTTATTACCGGGTAGGGGAGGCG
CTTTTCCCA AGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTG
GCCTCTGGCCTCGCACACATTCCACATCCCCCGGTAGGCGCCAACCGGCTCCGTTCTTTG
GTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGC
AGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAG
ATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGC
AGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCG
GGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCG
GCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGG
GCCTTTCGGAATTCATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGAC
GTCCCCCGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCA
CACCGTCGACCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCA
CGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGG
CGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCC
CGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCT
CCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCG
CCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCG
GCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTT
CTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGC
ACCTGGTGCATGACCCGCAAGCCCGGTGCCTGATAGAACTTGTTTATTGCAGCTTATAAT
GGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATT
CTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGATAACTTCGTATAATGT
ATGCTATACGAAGTTATGCGGCCGCAATCGTCGACCTGCAGGCATGCAAGCTTATTGATTA
TTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTT
CCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCC
ATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGT
CAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGC
CAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGT
ACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACC
ATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCC
CAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGG
GGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCG
GAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCG
AGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCT
GCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCT
CTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCT
GTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTG
AGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTG
TGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCT
GCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCC
GGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGG
GGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACC
CCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCC
GTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGG
GTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCG
GCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGTCGCAGCCATTGCCTTTT
ATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGTGGAGCCGA
AATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCG
CCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCC
TTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGGCGGCTGCCTTCGGGGGGGACG
GGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAAC
CATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGT
CTCATCATTTTGGCAAAGAATTCCTCGACCTCGAGATGACAGAGTTACCTGCACCGTTGTCCTACTTCCAGAATGCA CAGATGTCTGAGGACAACCACCTGAGCAATACTGTACGTAGCCAGAATGACAATAGAGAACGGCAGGAGCACAACGA CAGACGGAGCCTTGGCCACCCTGAGCCATTATCTAATGGACGACCCCAGGGTAACTCCCGGCAGGTGGTGGAGCAAG ATGAGGAAGAAGATGAGGAGCTGACATTGAAATATGGCGCCAAGCATGTGATCATGCTCTTTGTCCCTGTGACTCTC TGCATGGTGGTGGTCGTGGCTACCATTAAGTCAGTCAGCTTTTATACCCGGAAGGATGGGCAGCTAATCTATACCCC ATTCACAGAAGATACCGAGACTGTGGGCCAGAGAGCCCTGCACTCAATTCTGAATGCTGCCATCATGATCAGTGTCA TTGTTGTCATGACTATCCTCCTGGTGGTTCTGTATAAATACAGGTGCTATAAGGTCATCCATGCCTGGCTTATTATA TCATCTCTATTGTTGCTGTTCTTTTTTTCATTCATTTACTTGGGGGAAGTGTTTAAAACCTATAACGTTGCTGTGGA CTACATTACTGTTGCACTCCTGATCTGGAATTTTGGTGTGGTGGGAATGATTTCCATTCACTGGAAAGGTCCACTTC GACTCCAGCAGGCATATCTCATTATGATTAGTGCCCTCATGGCCCTGGTGTTTATCAAGTACCTCCCTGAATGGACT GCGTGGCTCATCTTGGCTGTGATTTCAGTATATGATTTAGTGGCTGTTTTGTGTCCGAAAGGTCCACTTCGTATGCT GGTTGAAACAGCTCAGGAGAGAAATGAAACGCTTTTTCCAGCTCTCATTTACTCCTGCACAGAAAGGGAGTCACAAG ACACTGTTGCAGAGAATGATGATGGCGGGTTCAGTGAGGAATGGGAAGCCCAGAGGGACAGTCATCTAGGGCCTCAT CGCTCTACACCTGAGTCACGAGCTGCTGTCCAGGAACTTTCCAGCAGTATCCTCGCTGGTGAAGACCCAGAGGAAAG GG GAGTAAAACTTGGATTGGGAGATTTCATTTTCTACAGTGTTCTGGTTGGTAAAGCCTCAGCAACAGCCAGTGGAGACTGGAACACAACCATAGCCTGTTTCGTAGCCATATTAATTGGTTTGTGCCTTACATTATTA CTCCTTGCCATTTTCAAGAAAGCATTGCCAGCTCTTCCAATCTCCATCACCTTTGGGCTTGTTTTCTACTTTGCCAC AGATTATCTTGTACAGCCTTTTATGGACCAATTAGCATTCCATCAATTTTATATCTAGAGATCTGGCAGCGGAGAGG
GCAGAGGAAGTCT TCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGTTCGAAATGGTGAGCAAG
GGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAA
ACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCT
GACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGA
CCACCTTGACCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCAC
GACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAG
GACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTG
AACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACA
AGCTGGAGTACAACTACAACAGCCACAAGGTCTATATCACCGCCGACAAGCAGAAGAAC
GGCATCAAGGTGAACTTCAAGACCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCG
CCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAAC
CACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACAT
GGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACA
AGTGAGAGCTCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCC
GTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAA
ATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGA
CAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCGATCG。
The sequence of 5' ends junction between Insert Fragment and host genome is expressed as：
ACCCCACAGTGGGGCAAGCTTGGATCCTC and its complementary series.
The sequence of 3' ends junction between Insert Fragment and host genome is expressed as：
CGATCGGCGGCCGCTACTAGGGACAGGATT and its complementary series.
In another embodiment, the nucleotide sequence of insertion can include any variant and/or PS1 of coding BACE1
Any variant sequence.Variant includes allele variant (such as polymorphism), the alternative splice forms of such as Different Individual
The naturally occurring variant Deng caused by.Variant preferably substantially with sequence homology according to the present invention, that is, is shown with the present invention's
Sequence typically at least about 80%, preferably at least about 90%, more preferably at least about 95% nucleotide sequence homology.The present invention
The variant of gene is also included within the nucleotide sequence with sequence as defined above (or its complementary strand) hybridization under stringent hybridization condition.
Typical stringent hybridization condition includes temperature higher than 42 DEG C, and salinity is equal to or less than 200mM.
Drug screening method
In one embodiment, the present invention relates to the hiPSC cell line selections use created by using the method for the present invention
In the method for the therapeutic agent for the treatment of AD.This method can include following multiple steps：HiPSC cell lines are divided into function again
Nerve member；Medical compounds is administered in neuronal culture；In one section of developing approach in the presence of medical compounds
Between；And measure beta-secretase level, A β -40 concentration, A β -42 concentration and A β 42/A β -40 compare etc..
Beta-secretase level of the present invention can be detected by this field conventional method in rna level and protein level.
In a detailed embodiment, being overexpressed the hiPSC cell lines of BACE1 genes will be used to generate substantial amounts of work(
It can nerve member.Neuronal cell from hiPSC cell lines will be cultivated in 96 orifice plates.Compound to be screened is added
Into neuronal culture 2 days to 2 weeks.Compound will put down the effect for reducing β-secretion enzyme level and/or A β -42 peptides
Row measurement.
In another embodiment, the hiPSC cell lines of PS1dE9 genes are overexpressed for generating substantial amounts of work(
It can nerve member.Neuronal cell from hiPSC cell lines is cultivated in 96 orifice plates.Compound to be screened is added to
2 days to 2 weeks in neuronal culture.Measure effect of the compound to A β -42 peptide reductions.
In another embodiment, using the BACE1 genes being overexpressed at AAVS1 sites any variant and/or
The hiPSC cell lines of any variant of PS1 genes are first to generate substantial amounts of functional nerve.Variant includes such as Different Individual
Allele variant (such as polymorphism), naturally occurring variant caused by optional splicing form etc..Variant preferably substantially with
Sequence homology according to the present invention, that is, show with the present invention sequence typically at least about 80%, preferably at least about 90%, more
Preferably at least about 95% nucleotide sequence homology.The variant of gene of the present invention be also included under stringent hybridization condition with such as
The nucleotide sequence of sequence (or its complementary strand) hybridization of upper definition.Typical stringent hybridization condition includes temperature higher than 42 DEG C,
Salinity is equal to or less than 200mM.
The embodiment being described with reference to the drawings in the application is explanatory, illustrative, and for usually understanding
The present invention.Embodiment should not be construed as the limitation present invention.In entire description, the same or similar element and with identical
Or the element of identity function is indicated by the same numbers.It will also be understood that term used in this application is only used for describing
The purpose of particular implementation, is not intended to limit, because the scope of the present invention only is limited by the following claims.
Embodiment
The purpose that embodiment is merely to illustrate is provided.
Embodiment 1：Generate parent's hiPSC cell lines for genome editor
Two hiPSC cell lines, UCIS3007 and IPSN0041 are generated, and as parent's hiPSC cell lines, is used for
The genes lesion hiPSC cell lines such as preparation.UCIS3007 derives from the urine cell of non-diseased women donor, and the urine cell leads to
The reprogramming method for crossing retrovirus-mediated is generated using four kinds of transcription factors (Oct4, Sox1, Klf4 and cMyc).
Donor urine epithelial cell is cultivated using urine epithelial cell proliferation culture medium (CIB, catalog number (Cat.No.) UC-0302).It is previous in virus infection
My god, will about 20,000 urine epithelial cell is inoculated into 12 orifice plates.The cell transfected with retroviral vector is used
HiPSC reprogramming blood serum mediums (CIB, catalog number (Cat.No.) RE-0201) are cultivated.At the 6th day, seed cells into thin containing raising
It is handled on the T25 flasks of born of the same parents and with mitomycin C.By about 10,000 cell inoculations in each T25 flasks and with again
Programming culture medium is cultivated about 20 days together, until there are hiPSC clones.Then, by clone's picking to Matrigel
It is used to be further purified and expand on (Corning, catalog number (Cat.No.) 352477) coated plate.
IPSN0041 derives from the umbilical cord matrix cells of non-diseased donor, and the umbilical cord matrix cells pass through no footprint side
Method (footprint-free method) is generated using episomal vector of the tool there are six transcription factor.By about 1,000,000 navels
Tape base cell plastid is mixed with nuclear transfer reagent (Lonza, catalog number (Cat.No.) VPI-1005) using Amaxa Nucleofector kits II
The additional plasmid (3 μ gpCEP4-EO2S-EN2K, 2.4 μ gpCEP4-M2L, 3.2 μ gpCEP4-EO2S-1) of conjunction carries out electricity together
Transfer.The cell of transfection is inoculated into immediately with the coated two T25 flasks of Matrigel (Corning, catalog number (Cat.No.) 352477)
In, it is cultivated using hiPSC reprogramming culture mediums (CIB, catalog number (Cat.No.) RE-0202).The 15th day after transfection, culture medium is changed to
In addition mTeSR1 is cultivated 7 days, until there are hiPSC bacterium colonies.Clone of the selection with typical case's hiPSC forms is for further pure
Change and expand.
The identification of hiPSC clones follows international standard43-44, including foreign gene silence and pluripotency marker's detection of expression,
Exogenous origin gene integrator measure, the analysis of promoter demethylation, karyotyping experiment, differentiation potential experiment (embryoid is formed), abnormal
(Fig. 1) such as tire knurl are tested and cell ID (STR) is tested.In addition, before genome editing is carried out, both are also verified
The Neural lineage differentiation potential (Fig. 2) of parent's hiPSC cell lines.
Embodiment 2：Build donor vehicle and targeting vector
HiPSC genetic engineering transformations are carried out using most common genome editing technique CRISPR/Cas9 systems.For
AD related genes targeted integration builds the specific C RISPR for targeting AAVS1 sites to safe port site (AAVS1 sites)
SgRNA carriers.SgRNA designs and builds the method followed described in document29.The target site of BpiI enzymes is located on carrier, identification
Site is located at cut portion.Therefore, target site intervening sequence can be digested by a step and is cloned into carrier.After digestion, carry
Body contains the cohesive end of 5'GTGG3' and 5'GTTT3'.By synthesize CACC+ target intervening sequences cohesive end and
The sticky terminal of AAAC+ reverse complementary sequences, sgRNA become the Double stranded oligonucleotide acid sequence with additional cohesive end,
It may be connected to cas9 carriers.CRISPR-cas9 plasmids are usually built by two-step method.The first step is the few nucleosides of processing synthesis
The cleavage site of acid sequence.Second step is by the oligonucleotide sequence of processing and the CRISPR- by limiting collagenase treatment
Cas9 carriers connect.
It builds, generates first comprising PGK-PURO-SV40PA, P2A-EGFP-BGHPA and CAG promoter for donor
Skeleton carrier and multiple cloning sites (MSC).In order to build final donor, 5 kinds of plasmids are generated：1)T-HindIII-CAG-
EcoRI；2)T-SalI-P2A-EGFP-BGHPA-NotI；3)T- HindⅢ-PGK-PURO-SV40PA-HindIII；4) synthesize
PUC19-EcoRI-BACE1cds-SalI；With 5) PZD carriers (Sigma-Aldrich).The first step be digested plasmid 1,2,
4 and 5, recycle four segments, HindIII-CAG-EcoRI- ,-LALI-EGA-EGFP-BGHPA-NotI-, EcoRI-
Then BACE1cds-SalI- and HindIII-PZDonor-HindIII- link together all segments.It is thin to convert DH5 α
After born of the same parents, by the intermediate carrier that positive clone identification is PCBEB6.It is digested by HindIII, then connection PCB EB6 and T-
HindIII-PGK-PURO-SV40PA-HindIII generates final donor vehicle.The donor vehicle is named as CIB-PCBEB
(Fig. 3 A).By the way that in gene order of the both ends synthesis with BglII and XhoI cleavage sites, completion AAVS1 sites are overexpressed
All foreign genes.Target gene is inserted into carrier by using BglII and XhoI digested vectors, then by Insert Fragment and institute
State carrier connection.Then final donor plasmid passes through sequence verification for converting DH5 α cells.BACE1 genes and PS1dE9 bases
Because the donor of overexpression is built as shown in figs. 3 b and 3 c.
Embodiment 3:In AAVS1 sites targeted integration AD related genes
In order to which AD related genes are introduced AAVS1 sites, by parent hiPSC mTeSR1 (Stemcell
Technologies, catalog number (Cat.No.) 05850) coating Matrigel (Corning, catalog number (Cat.No.) 354277) 6 orifice plates on 37
DEG C, 5%CO2Lower culture.Converge harvest cell with 80%, and pass through electroporation (Amaxa Nucleofector II, journey
Sequence A-024) and Cas9-sgRNA plasmids and donor plasmid cotransfection.The cell number of each transfection reaction is 0.5 to 1 × 106,
Quantity with plasmid is：4 μ g of 2.5 μ g of Cas9-sgRNA and donor plasmid.The cell of transfection is inoculated into use immediately
In coated six orifice plates of Matrigel, it is incubated with mTeSR1 and 10 μM of Y-27632 (Sigma, catalog number (Cat.No.) Y0503).It is incubated
48 it is small when after, it is small that the puromycin (Xiya Reagent, catalog number (Cat.No.) 1014553) of 0.5 μ g/ml is added in culture medium 24
When be used for drug screening.Single cell clone is prepared by the following procedure：Chloramphenicol resistance cell is connect with the density of 1000 cell/ml
It plants onto the coated T25 flasks of Matrigel, is incubated with mTeSR1 and 10uM Y-27632.After 10 days, chosen with microscope auxiliary
Single cell clone is taken, is then transferred into coated 96 orifice plates of Matrigel.Each clone is divided into two equal portions, and two
It is cultivated on a 96 orifice plate, one is used to breed, and one is used to identify.In order to verify gene integration, pass through DNA lytic reagent boxes
(QuickExtract TMDNA Extraction Solution 1.0, Epicenter, catalog number (Cat.No.) QE09050) cell lysis,
Then by connecting PCR identifications, there are Insert Fragment (Fig. 4 B).By connecting the positive colony of PCR identifications, by being sequenced into one
Step demonstrate,proves (Fig. 4 C).In addition, the clone of selection also carries out versatility test and karyotyping to eliminate by single cell clone process
The variation of introducing.Two hiPSC cell lines of composition overexpression BACE1 and PS1dE9 genes are generated using similar strategy
(Fig. 4 A and Fig. 5 A).
Embodiment 4：It is extensive to generate nerve cell
As described above, can conformably and repeatably generate largely with the relevant human neures of AD, it is used for for foundation
The physiological cells platform of drug screening is vital.In current technology, hiPSC is divided into functional nerve member and relates to
And interminable process, and it is subjected to a series of variation of condition of culture.Therefore, hiPSC is directly inoculated in 96 orifice plates to generate
Neuron causes to make a variation between the hole in terms of the quality and quantity of huge differentiated neuron.This huge variation is not suitable for
Drug screening.In order to solve this problem, step-by-step movement flow (Step-wise process) is developed to control variation.The first step
It is the neural stem cell (NSC) that high quality is generated from hiPSC.The step makes the whole process from hiPSC to mature neuron
Shorten 6-8 weeks.Second step is that NSC is divided into neural progenitor cell (NPC), is further reduced point for generating mature neuron
Change the time.In order to generate NSC original seeds, hiPSC is seeded in containing Neuronal induction media (Stemcell
Technologies, catalog number (Cat.No.) 05835) and 12 orifice plates of 10uM Y-27632 (Sigma, catalog number (Cat.No.) Y0503) on.After 5 days,
The embryoid ball of formation is laid on coated 12 orifice plates of Matrigel, in 37 DEG C of CO2It is incubated in incubator, and it is every
It replaces Neuronal induction media.After 7 days, picking NSC sample rosette cells, and be transferred in 24 orifice plates.Culture medium is used
NSC proliferated culture mediums (CIB, catalog number (Cat.No.) NE-0603) replace.After 7 to 8 days, picking NSC like cells and 48 orifice plates are transferred to
In.When NSC density, which reaches 90%, to be converged, it is transferred into 6 orifice plates further to expand and store.In order to generate work(
Can nerve member, will about 8 × 104/cm2NSC be transferred to and be coated with poly- L-Orn (Sigma, Cat.) and layer No.P4957
In 6 orifice plates of Fibronectin (Sigma, catalog number (Cat.No.) 2020), and in Neuronal induction culture medium (Stemcell
Technologies, catalog number (Cat.No.) 08500) in culture.After 7 days, culture medium is changed to neuronal maturation culture medium
(Stemcell Technologies, catalog number (Cat.No.) 08510) continues 2 to 3 week of culture.Pass through immunostaining neuronal specificity
Biomarker such as Tuj1 confirms mature neuron (Fig. 6).
Embodiment 5：The beta-secretase being overexpressed in modified hiPSC cell lines
As described above, generate two hiPSC cell lines using the carrier created by the present invention.One hiPSC cell line
(IPSN0041-21) BACE1 (beta-secretase 1) gene of constructive expression is contained in AAVS1 sites.It is for this, it will
BACE1 genes are in the expression of RNA and protein level and its grade genes parental department (IPSN0041) in RNA and protein level
Expression be compared.In order to which quantitative BACE1 mRNA are expressed, total RNA is extracted with TRIzol (Sigma, catalog number (Cat.No.) T9424).
It is carried out using 7500 systems of ABITM and fluorescent dye SYBR Premix EXTaqTMII (TaKaRa, catalog number (Cat.No.) RR820A)
qPCR.Housekeeping gene beta-actin is used as reference, and each data point is to repeat average value three times.The results show that with
IPSN0041 is compared, the BACE1 mRNA of IPSN0041-21 expression much higher, table in iPSC and neuron from iPSC
The bright BACE1 genes being transferred to are really overexpression (Fig. 7 A) in IPSN0041-21.The expression of BACE1 albumen uses ELISA
Kit (Thermo Fisher, catalog number (Cat.No.) P0013) carries out.NSC and mature neuron are obtained using the above method, passes through egg
White matter lysis buffer (Biyuntian Biotechnology, catalog number (Cat.No.) P0013) cell lysis, and tried using ELISA
Agent box measures BACE1 protein concentrations according to the manufacturer's instructions.Similar to RNA as a result, BACE1 eggs in IPSN0041-21
(beta-secretase 1) the horizontal BACE1 albumen water for being significantly higher than parental department IPSN0041 in the NSC and neuron from hiPSC in vain
Flat (Fig. 7 B).
Embodiment 6：Express A β -42 peptides in modified hiPSC cell lines
For A β -42 peptides detect, by mature neuron neuronal maturation culture medium (Stemcell Technologies,
Catalog number (Cat.No.) 08510) in culture at most 7 weeks.It was collected supernatant with 1 week for interval.User/rat A β 42ELISA kits
(WAKO, catalog number (Cat.No.) 290-62601) measures the concentration of A β -42 peptides.In order to standardize, the total protein collected every time is also measured
(Thermo-Fisher, Pierce BCA Protein Assay Kit, catalog number (Cat.No.) 23225).As expected, observe
The expression quantity of A β -42 peptides is expressed into IPSN0041-21 neuronal cultures higher than expression A β -42 in IPSN0041 neurons
The expression quantity (Fig. 8 A) of peptide.It is through six weeks further study showed that, compared with the neuron from IPSN0041, come from
The neuron of IPSN0041-21 has different expression patterns.A β -42 peptides in IPSN0041 neurons (wild type)
Level is very low in neuron culture first week, increases as the time elapses, peaks within the 5th week, decline in the 6th week；And
A β -42 expression in IPSN0041-21 neurons is high in neuron culture the last fortnight, reduces (Fig. 8 B) over time.
Due to the ageing process of 6 weeks simulation mature neurons of extracorporeal neuron cell culture, observed result shows for wild
Type neuron, A β -42 levels increase during neuron senescence, this is consistent with the disease mechanisms of AD.A β -42 are the 6th
The decline in week may be due to the neuronal death in culture medium.On the other hand, A β -42 in early stage IPSN0041-21 neurons
High level expression can be construed to the overexpression of BACE1 transgenosis, the reduction that A β -42 are expressed after 3 weeks is due to refreshing before maturation
Through meta function obstacle or since β-secretase excessively causes the neuronal death.The observed result shows that beta-secretase is expressed
Rise is harmful to neuronal survival.
Embodiment 7：Use modified hiPSC cell line selections medical compounds
Substantial amounts of neuron progenitor cell (NPC) is generated from modified hiPSC cell lines IPSN0041-21.In order to
Screening compound measure is carried out, NPC is seeded in 96 orifice plates of the cell density for 50,000 cells/well.NPC is in nerve
Culture at most 4 weeks in first maturation medium (Stemcell Technologies, catalog number (Cat.No.) 08510).It was received with one week for interval
Collect supernatant.User/rat A β 42ELISA kits (WAKO, catalog number (Cat.No.) 290-62601) were surveyed two weeks, three weeks or surrounding
Measure A β -40 and A β -42 concentration.Compound to be tested adds in neuronal culture in measurement A β -40 and A β -42 peptide the last weeks
In.As shown in figure 9, the influence that two kinds of new compounds (B3 and B16) of test generate A β peptide.The results show that B3 and B16 subtract
The generation of few A β -40 and A β -42 peptides, but the ratio of A β -42 and A β -40 are not influenced.It attracts people's attention, with B3 and B16 processing
Situation compare, positive control (common beta-secretase inhibitor (Sigma-Aldrich, catalog number (Cat.No.) S4562)) is disproportionate
Ground reduces the generation of A β -42 peptides and A β -40 peptides, as shown in the ratio of higher A β -42 and A β -40.Should the result shows that, due to
The overexpression of BACE1 genes causes the expression of the A β peptide in IPSN0041-21 to raise, and improves A β -40 and A β -42 peptides really
The sensitivity of measure, so that influence of the potential drug compound to different type A β peptide is distinguished.Particularly, this is
System can screen the more effective compound for reducing A β -42 and producing.
Conclusion
Present invention description is overexpressed by the AD related genes in hiPSC creates the relevant AD cell models of physiology, institute
State AD related genes such as BACE1 and PS1 and its variant.The donor vehicle of several uniquenesses is generated, for expeditiously existing
Safe port site (AAVS1) targeted integration AD related genes in human genome.Targeted integration AD is carried out in AAVS1 sites
Related gene provides safety and controlled transgene expression, the shortcomings that overcoming common random integration method, such as unknown copy
Number and the Latent destruction to endogenous gene.The hiPSC cell lines generated using these donor vehicles show high-caliber β-
Secretase activity and/or high-caliber A β -42 peptides, A β -42 peptides are the main components that amyloid plaque is formed.This hair
It is bright to establish new raji cell assay Raji platform, it carries out being overexpressed AD dependency basis using the neuronal cell from hiPSC cell lines
Cause.The preliminary screening of potential drug candidate compound shows that compared with the currently available technology of this field the present invention creates thin
Born of the same parents measure platform and provide clear superiority to screen new β-Secretase inhibitors and/or A β -42 inhibitor peptides.
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Claims (10)
1. a kind of method for the cell model for preparing Alzheimer disease (AD), including AD related genes are integrated into hiPSC
In to induce the beta-secretase of hiPSC is horizontal to increase and/or A β -42 peptides increase.
2. composition is overexpressed in hiPSC the method for claim 1 wherein the AD related genes.
3. the method for claim 1 or 2, wherein the AD related genes are the mutation app gene or mutation PS that AD is caused to fall ill
Gene.
4. the method for claim 3, wherein the mutation PS genes are PS1dE9 genes.
5. the method for claim 1 or 2, wherein the AD related genes are BACE1 genes.
6. the method for any one of claim 1-5, wherein the AD related genes be selected from AD is caused to fall ill mutation app gene,
PS1dE9 genes and BACE1 genes.
7. the method for any one of claim 1-6, wherein the AD related genes are integrated by way of locus specificity
In hiPSC.
8. the method for claim 7, wherein the AD related genes in AAVS1 integrations into hiPSC.
9. the cell model for passing through the Alzheimer disease (AD) that the method for any one of claim 1-8 generates.
10. a kind of method for modifying hiPSC, including importing AD related genes to hiPSC so that its composition is overexpressed.
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| WO2017079673A1 (en) * | 2015-11-04 | 2017-05-11 | Fate Therapeutics, Inc. | Genomic engineering of pluripotent cells |
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| WO2017079673A1 (en) * | 2015-11-04 | 2017-05-11 | Fate Therapeutics, Inc. | Genomic engineering of pluripotent cells |
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| CN112899280A (en) * | 2021-04-09 | 2021-06-04 | 中国药科大学 | AD cell model established based on CRISPR/Cas9 gene editing technology and construction method and application thereof |
| CN112899280B (en) * | 2021-04-09 | 2023-11-03 | 中国药科大学 | AD cell model established based on CRISPR/Cas9 gene editing technology, and construction method and application thereof |
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