CN112695032A - Promoter pLRRK2 and application thereof - Google Patents

Promoter pLRRK2 and application thereof Download PDF

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CN112695032A
CN112695032A CN202011434481.9A CN202011434481A CN112695032A CN 112695032 A CN112695032 A CN 112695032A CN 202011434481 A CN202011434481 A CN 202011434481A CN 112695032 A CN112695032 A CN 112695032A
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recombinant adeno
plrrk2
promoter
plrrk
associated virus
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CN112695032B (en
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林剑邦
陈晔菲
路中华
杨立新
朱钰媛
徐冬冬
李梦奇
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Shenzhen Institute of Advanced Technology of CAS
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Abstract

The invention discloses a promoter pLRRK2 and application thereof, wherein the nucleotide sequence of the promoter pLRRK2 is shown as SEQ ID NO: 1 is shown. The promoter pLRRK2 can be applied to recombinant adeno-associated virus, so that exogenous genes can be efficiently expressed in neocortex, and the promoter has wide application prospect in the neural loop tracing field and the gene therapy field.

Description

Promoter pLRRK2 and application thereof
Technical Field
The invention belongs to the technical field of nerve engineering, and particularly relates to a promoter pLRRK2 and application thereof.
Background
The neocortex is a part of the mammalian central nervous system and is involved in a variety of important functions including sensation, perception, voluntary movement, learning, cognition, and the like. The analysis of the neural circuits connecting the neocortex to the respective nuclei can help us to better understand the higher functions of the brain. The research on neocortex can help us to understand the pathogenesis of cortical-related neurological diseases and further search a treatment method thereof. However, there is no viral tool available to efficiently label neocortex, which has hampered neocortex studies.
Adeno-associated virus is a single-stranded DNA virus without an envelope. The recombinant adeno-associated virus (rAAV) is a recombinant virus which is reconstructed on the basis of wild AAV. The recombinant adeno-associated virus has become a virus tool widely used in the field of neuroscience due to the advantages of long-term stable expression of mediated genes in mammals, low immunogenicity, wide host range and the like, and is also a gene therapy vector tool most widely applied in clinical experiments of central nervous system diseases at present.
In the process of applying rAAV to neuroscience research and clinical treatment, the key point of gene delivery is how to improve the space-time specificity and the expression level of gene expression. Strategies to regulate foreign gene expression directly affect the efficiency and safety of gene delivery. For neuroscience research, to achieve labeling of specific neurons in a particular neural circuit, different brain regions or nuclei are labeled. For gene therapy of nervous system diseases, the manipulation of neurons in the whole brain or in different subtypes is required to intervene in different nervous system diseases. One important approach is to select rAAV viruses of different serotypes. Among the presently available reports, there are AAV serotypes suitable for use in the nervous system: type 1, type 2, type 5, type 6, type 8, type 9, DJ, PHP.B, PHP.eB, PHP.S, Retro, rh10, etc. Different serotypes of viruses have different infection efficiencies and spreading capacities for different brain regions, and therefore, different rAAV serotypes can be selected as gene delivery vehicles according to the subject or target of treatment. However, due to the complexity of the central nervous system, these serotypes are far from meeting the current requirements for gene delivery specificity. Another approach is to use tissue-specific promoters in the rAAV genome. Promoters are cis-acting elements essential in the regulation of gene expression. Under the control of promoters with expression specificity, exogenous genes are generally expressed only in certain specific cell types or brain regions. In the rAAV, the specific promoter is used for driving gene expression, so that the gene expression efficiency can be improved, and the immunoreaction generated in the rAAV infection process is reduced.
Disclosure of Invention
In order to solve the problems of the background art, the invention aims to provide a neocortex high-efficiency expression promoter pLRRK2 and application thereof. The promoter pLRRK2 can be applied to recombinant adeno-associated virus, so that exogenous genes can be efficiently expressed in neocortex, and the promoter has wide application prospect in the neural loop tracing field and the gene therapy field.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: in one aspect, the invention provides a promoter pLRRK2, wherein the nucleotide sequence of the promoter pLRRK2 is as shown in SEQ ID NO: 1 is shown.
Further, the promoter pLRRK2 is a partial sequence of the human leucine-rich repeat kinase 2(LRRK2) gene, a sequence which is 2201bp near the transcription initiation site of the LRRK2 gene is selected, and an ATG sequence contained in the sequence is replaced by a TTG sequence to be used as a final promoter sequence.
Further, the promoter plrrrk 2 was expressed efficiently in neocortex.
In another aspect, the present invention provides a recombinant adeno-associated virus vector comprising the promoter described above.
Further, the recombinant adeno-associated viral vector comprises Paav-pLRRK 2-fluorescent protein, Paav-pLRRK 2-functional protein or Paav-pLRRK 2-therapeutic protein.
Further, the fluorescent protein includes eYFP, tdTomato;
preferably, the functional protein comprises a optogenetically-related protein or a chemically genetically-related protein.
The Paav-pLRRK 2-fluorescent protein (fluorescent protein including eYFP, tdTomato) can realize the nerve loop multicolor tracing. Paav-pLRRK 2-functional proteins (including optogenetically related proteins or chemogenetically related proteins) can make recombinant adeno-associated viruses that activate or inhibit specific neurons. The Paav-pLRRK 2-therapeutic protein can be used in gene therapy.
Further, the recombinant adeno-associated virus vector Paav-pLRRK2-eYFP adopts Paav-hSyn-eYFP as a skeleton vector, hSyn is replaced in the vector to be a pLRRK2 promoter, and the recombinant adeno-associated virus vector Paav-pLRRK2-eYFP is obtained.
Further, the preparation method of the recombinant adeno-associated virus vector Paav-pLRRK2-eYFP comprises the following steps: cloning a promoter pLRRK2 into a PUC-57 vector to obtain a PUC57-pLRRK2 vector; treating the Paav-hSyn-eYFP vector by using restriction endonucleases xbalI-HF and kpnI-HF, and treating the PUC57-pLRRK2 vector by using the restriction endonucleases xbalI-HF and kpnI-HF; and (3) recovering, purifying and connecting the two enzyme digestion products to obtain the recombinant adeno-associated virus vector Paav-pLRRK 2-eYFP.
In another aspect, the present invention provides a recombinant adeno-associated virus comprising any one of the recombinant adeno-associated virus vectors described above.
Further, the recombinant adeno-associated virus comprises AAVDJ-retro-pLRRK 2-fluorescent protein, AAVDJ-retro-pLRRK 2-functional protein or AAVDJ-retro-pLRRK 2-therapeutic protein;
preferably, the fluorescent protein comprises eYFP, tdTomato;
preferably, the functional protein comprises a optogenetically-related protein or a chemically genetically-related protein.
The recombinant adeno-associated virus AAVDJ-retro-pLRRK2-eYFP and AAVDJ-retro-pLRRK2-tdTomato can effectively reverse and cross synapse to mark neural circuits from the nigra reticular part to the cortex and express eYFP or tdTomato with high fluorescence intensity.
In still another aspect, the present invention provides a kit comprising the promoter plrrrk 2 described above, the recombinant adeno-associated vector described above, or the recombinant adeno-associated virus described above.
In still another aspect, the present invention provides a use of the promoter plrrrk 2, the recombinant adeno-associated vector, the recombinant adeno-associated virus, or the kit in the preparation of a neural circuit tracing and/or manipulating neural cells and/or gene therapy reagents.
In a further aspect, the invention provides a neural network tracer comprising Paav-pLRRK 2-fluorescent protein and/or a recombinant adeno-associated virus comprising Paav-pLRRK 2-fluorescent protein;
preferably, the fluorescent protein comprises eYFP, tdTomato;
preferably, the recombinant adeno-associated virus comprises AAVDJ-retro-pLRRK2-eYFP, AAVDJ-retro-pLRRK 2-tdTomato.
In a further aspect, the invention provides a use of the neural network tracer described above in tracing a nigral reticular compartment-cortex loop.
Compared with the prior art, the invention has the following beneficial effects:
(1) the promoter pLRRK2 has high-efficiency expression efficiency in neocortex, can realize high-efficiency expression of exogenous genes in neocortex, improves the expression specificity after virus infection, and has wide application prospect in the fields of neural loop tracing and gene therapy;
(2) the promoter pLRRK2 of the invention has no cytotoxicity in bacteria and mammalian cells;
(3) the recombinant adeno-associated virus vector adopts pLRRK2 as a promoter, and has high-efficiency expression efficiency in neocortex;
(4) the recombinant adeno-associated virus can efficiently and reversely mark the neural circuit from the substantia nigra compacta to the cortex in the brain, and has high marking efficiency and strong infection capacity.
Drawings
FIG. 1 is a flow chart of the construction of the Paav-pLRRK2-eYFP vector of the present invention;
FIG. 2 is a graph showing the expression efficiency of AAVDJ-retro-pLRRK2-eYFP according to the present invention in the black reticulum and cortex.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
EXAMPLE 1 construction of the pUC57-pLRRK2 vector
The pLRRK2 promoter is a partial sequence of a human leucine-rich repeat kinase 2(LRRK2) gene, a sequence which is 2201bp near the transcription initiation site of the LRRK2 gene is selected, an ATG sequence contained in the sequence is replaced by a TTG sequence to be used as a final promoter sequence, and the nucleotide sequence of the promoter pLRRK2 is shown as SEQ ID NO: 1 is shown.
The promoter pLRRK2 is artificially synthesized and then cloned in a PUC-57 vector to obtain a PUC57-pLRRK2 vector.
Example 2 construction of recombinant adeno-associated Virus vector Paav-pLRRK2-eYFP
In this example, because YFP is used as a reporter gene of a promoter, when constructing a recombinant vector, a recombinant adeno-associated vector Paav-hSyn-eYFP containing YFP gene is selected as a vector backbone to link with a pLRRK2 promoter, and the steps are as follows:
as shown in FIG. 1, Paav-hSyn-eYFP vector is treated by using restriction enzymes xbalI-HF and kpnI-HF, and PUC57-pLRRK2 vector is treated by using restriction enzymes xbalI-HF and kpnI-HF, and then enzyme digestion is carried out for 3h at 37 ℃, wherein the enzyme digestion system is shown in tables 1 and 2, the enzyme digestion product is recovered and then linked by using a linking premix (2 × ligation premix, TAKARA) at 16 ℃ for 30min, and the system is shown in Table 3, thus obtaining the successfully linked Paav-hSyn-eYFP vector.
TABLE 1 Paav-hSyn-Eyfp vector restriction enzyme System
Reagent Dosage of
Enzyme digestion Buffer solution 10 XCutSmart Buffer 5μL
XbaI-HF 1μL
KpnI-HF 1μL
Paav-hSyn-Eyfp 1μg
ddH2O Make up to 50 μ L
TABLE 2 pUC57-pLRRK2 vector restriction enzyme system
Figure RE-GDA0002984714210000041
Figure RE-GDA0002984714210000051
TABLE 3 connection System
Reagent Dosage of
Connecting the premix 2 × ligation premix 5μL
Paav-Eyfp linking scaffold 0.5μL
pLRRK2 junction fragment 4.5μL
Example 3 preparation of recombinant adeno-associated Virus AAVDJretro-pLRRK2-eYFP
This example uses a three plasmid co-transfection method to prepare the virus. Before virus preparation, plasmids required for packaging virus are mentioned, including a recombinant adeno-associated virus vector Paav-pLRRK2-eYFP, a packaging plasmid AAVDJ-retro-RCB and a helper plasmid. The method comprises the following specific steps:
preparing cells: 293T cells were plated on plates containing whole medium (10% fetal bovine serum, 1% double antibody) and cultured in an incubator.
Preparation of transfection reagent: 5.25mL of ultrapure water, 75. mu.g of packaged plasmid, 75. mu.g of recombinant plasmid, 75. mu.g of helper plasmid and 800. mu.L of calcium chloride solution were aspirated and gently mixed. To the above reagent, an equal volume of 2 × HBS was added, vortexed, and allowed to stand for 30 min.
Transfection of cells: mu.L of chloroquine was added to each dish of cells, and the cells were incubated after addition of transfection reagent.
And (3) virus collection: after 72h, the transfected cells were collected in a centrifuge tube and centrifuged at 3000rpm for 30 min. The supernatant was discarded and cell lysate was added, followed by lysis of cells by repeated freeze-thaw. A cell disruption solution containing the virus was obtained.
And (3) purifying the virus: the cell disruption solution was centrifuged at 11500rpm for 30min, the supernatant was discarded and 200. mu.L of HBS was added thereto, followed by mixing, 200. mu.L of chloroform was added thereto, followed by centrifugation at 12000rpm for 5min, the supernatant was taken, 100. mu.L of 2.5mM NaCl and 100. mu.L of 40% PEG8000 were added thereto, followed by mixing by vortexing, and the mixture was cooled overnight in a refrigerator at 4 ℃. The overnight sample was centrifuged at 12000rpm for 30min, the supernatant was discarded, 30. mu.L of HBS and 0.5. mu.L of nuclease were added, and the mixture was allowed to stand for 30 min. Then 30. mu.L of chloroform was added and centrifuged at 12000rpm for 5 min. After purification, the product was stored in a freezer at-80 ℃.
And (3) determining the titer: the titer of the purified virus was measured by using AAV titer measurement dye-based fluorescent quantitation kit (TAKARA Co.) and found to be 1.0X 1013VG/mL。
Example 4 recombinant adeno-associated Virus labeling mouse substantia nigra reticula-cortical neural circuits
The purified recombinant adeno-associated virus was injected into bilateral substantia nigra reticulata (Bregma-3.40 mm; +/-1.40 mm; D:4.70mm) of mice by stereotactic injection. The recombinant adeno-associated viral capsid is AAVDJ-retro, has the ability to infect retrogradely and is capable of infecting neurons of the cortex upstream of the injected nigral reticulum. Three weeks later, mice were perfused to harvest brains, stained by immunohistochemical sectioning, and brain slice results were observed. The experimental results are shown in fig. 2, eYFP has high expression efficiency in mouse neocortex.
In conclusion, the invention is verified on mice, and the promoter pLRRK2 is proved to have high expression level in neocortex.
The above description is only a specific embodiment of the present invention, and not all embodiments, and any equivalent modifications of the technical solutions of the present invention, which are made by those skilled in the art through reading the present specification, are covered by the claims of the present invention.
SEQUENCE LISTING
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ctttggggtt atagacaccc atactcataa gtatttctga ataccaagag aagagtattt 60
aggtttgctt ctctcaactt ttcacctttc atttcatgta ccctgtcctt tgtctcagct 120
ctaatagctc tgagagctga ttacttttcg ggtgtcccaa gtatcaggat cctgcctagt 180
gcaactcaaa tttccaaaag ttaatttagt ggccttttgg tgaccagagc ttcagataac 240
tcacagggaa acaatgttta tttcctctcc cactaacagt cacaaaaaat cataaaaaag 300
agtagcgggg gcagttttga tggctaaccc ctctttccat cctttggggg aaaattgctc 360
atctccctat aggtggaact ctaaagacaa tgtattccta aaaggggcca tctgggcggt 420
gtcctctttt cccagcgccc tgatttctat tcttagatct ggagataggc ggctttcatt 480
tttcctgctc ccagttccca gaccttccgt ggggccgcag gatccccggc tggcgggtcg 540
cggagggtgg ccggccgggc tgcgcactgc gcgcctccgc tgcggggctc cgggcctgtg 600
gactcagcgg agtccgctga gtcagtttct tcccgcgcga ctcccggccg cgccgccgct 660
gcggtggaat ctggtcccag gaggcggcgt ccgcccgggg tccggtctag gcgtgcgtgg 720
gggccacggt cacggtcatc ccagccaggc ccggctccag cagccccacg gccgccgcca 780
gagttctgcg cggcccgtcg cctcggcgga gcctctggca ggcccctgag ctcgtttttg 840
gggcctgagt gggggaggag gaagccgagc aggagggctc cggagaggga gggcaacgcg 900
gggcggggag ctgcctcctt cctcataaac aggcgggcgt gggcgccgat ggggcccgcg 960
gggagcgctg gctgcgggcg gtgagctgag ctcgcccccg gggagctgtg gccggcgccc 1020
ctgccggttc cctgagcagc ggacgttctt gctgggaggg cggcgggttg gaagcaggtg 1080
ccaccttggc tagtggcagc tgtcaggggt gcgaagagga cgaggaaact ctgaagaagt 1140
tgatagtcag gctgaacatt gtccaggaag gaaaacagat agaaacgctg gtccaaatcc 1200
tggaggatct gctggtgttc acgtactccg agcgcggtaa tcacttgaaa ataaactgtg 1260
cttttatttt tgcaaacttt ctccccctcc ttacatttgc aaattttgtc ctcctcccct 1320
tgaccctgct caaacccgga ctcttaagga gccgcaaact cccatatcct ttccttaggg 1380
cagaaagcag ctgagaattt caggaaggtc ttcacctttt tgacttttct ccccgtttca 1440
gactaaaaag gagagggggt gctgtggatt gtgactttgc ttcttttccc cacccacttg 1500
ttttccagcc tccaagttat ttcaaggcaa aaatatcctt gtgcctctgt tgatcgtctt 1560
ggactcctat ttgagagtcg cgagtgtgca gcaggtaaag gcattgtttt cacttcaact 1620
cattctccct tctgtttgga aggagacgtt ttactggcaa tgttaatata gccgagagtt 1680
cttggttatt cccaaaattt ggcttgagga acctctgact gtgattttaa gatgggaata 1740
ttgttaaatc attacgcaat gtaaacggga tgaagagccc cagtatgtgt tccctgagtg 1800
tctttaagaa gtaactttat aaaaccaaca gtatggatgg tggtagaagg aggataaaaa 1860
tgggttcggt tttagtctcg ttattggcaa gatgaattca ttagtgttta gacttgacta 1920
ttccaagtat cttcccaata cagagcatgt cctagatgag aagattatga atagtttgga 1980
aaaggggaat aattaatagt gaagaatacg gcttgctttt gtttctgggt aagcatttta 2040
gggtatcatt ttgttgtata accattgttt acaagtgaga taagcatcta ttccactaag 2100
attgaagaga ttcatgtttg actgagtatg ctctattaac attctttaaa acatgtgaat 2160
atatgtcttt cttgttttca ggtgggttgg tcacttctgt g 2201

Claims (10)

1. The promoter pLRRK2, wherein the nucleotide sequence of the promoter pLRRK2 is shown as SEQ ID NO: 1 is shown.
2. The promoter pLRRK2 of claim 1, wherein the promoter pLRRK2 is efficiently expressed in neocortex.
3. A recombinant adeno-associated virus vector comprising the promoter according to claim 1 or 2.
4. The recombinant adeno-associated viral vector according to claim 3, wherein the recombinant adeno-associated viral vector comprises Paav-pLRRK 2-fluorescent protein, Paav-pLRRK 2-functional protein or Paav-pLRRK 2-therapeutic protein;
preferably, the fluorescent protein comprises eYFP, tdTomato;
preferably, the functional protein comprises a optogenetically-related protein or a chemically genetically-related protein.
5. A recombinant adeno-associated virus comprising the recombinant adeno-associated virus vector according to claim 3 or 4.
6. The recombinant adeno-associated virus of claim 5 wherein the recombinant adeno-associated virus comprises AAVDJ-retro-pLRRK 2-fluorescent protein, AAVDJ-retro-pLRRK 2-functional protein or AAVDJ-retro-pLRRK 2-therapeutic protein;
preferably, the fluorescent protein comprises eYFP, tdTomato;
preferably, the functional protein comprises a optogenetically-related protein or a chemically genetically-related protein.
7. A kit comprising the promoter pLRRK2 of any one of claims 1-2, the recombinant adeno-associated vector of any one of claims 3-4, or the recombinant adeno-associated virus of any one of claims 5-6.
8. Use of the promoter plrrrk 2 of any one of claims 1-2, the recombinant adeno-associated vector of any one of claims 3-4, the recombinant adeno-associated virus of any one of claims 5-6, or the kit of claim 7 for the preparation of a neural circuit tracking and/or manipulating neural cells and/or gene therapy reagents.
9. A neural network tracer comprising Paav-pLRRK 2-fluorescent protein and/or a recombinant adeno-associated virus comprising Paav-pLRRK 2-fluorescent protein;
preferably, the fluorescent protein comprises eYFP, tdTomato;
preferably, the recombinant adeno-associated virus comprises AAVDJ-retro-pLRRK2-eYFP, AAVDJ-retro-pLRRK 2-tdTomato.
10. Use of the neural network tracer of claim 9 in a nigral reticulum-cortex loop tracer.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007124096A2 (en) * 2006-04-21 2007-11-01 The Trustees Of Columbia University In The City Of New York Lrrk2 regulaton of neuronal process morphology
US20140100265A1 (en) * 2012-09-17 2014-04-10 The Trustees Of Columbia University In The City Of New York Gene Delivery Vehicles in the Treatment of Neurodegenerative Diseases
CN107630009A (en) * 2016-07-19 2018-01-26 中国科学院武汉物理与数学研究所 It is a kind of to be attenuated blast, replicate controllable HSV recombinant viruses and preparation method and application
US20190085394A1 (en) * 2015-12-14 2019-03-21 Parkinson's Institute Refining diagnosis and treatment of complex multi-symptom neurological disorders
CN111808862A (en) * 2020-07-30 2020-10-23 中国科学院精密测量科学与技术创新研究院 GABA (gamma-aminobutyric acid) energy neuron specific promoter and application thereof
CN112043710A (en) * 2020-09-28 2020-12-08 广州智睿医药科技有限公司 Medicine for treating or preventing diseases related to LRRK2 kinase or abnormal LRRK2 mutant kinase activity

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007124096A2 (en) * 2006-04-21 2007-11-01 The Trustees Of Columbia University In The City Of New York Lrrk2 regulaton of neuronal process morphology
US20140100265A1 (en) * 2012-09-17 2014-04-10 The Trustees Of Columbia University In The City Of New York Gene Delivery Vehicles in the Treatment of Neurodegenerative Diseases
US20190085394A1 (en) * 2015-12-14 2019-03-21 Parkinson's Institute Refining diagnosis and treatment of complex multi-symptom neurological disorders
CN107630009A (en) * 2016-07-19 2018-01-26 中国科学院武汉物理与数学研究所 It is a kind of to be attenuated blast, replicate controllable HSV recombinant viruses and preparation method and application
CN111808862A (en) * 2020-07-30 2020-10-23 中国科学院精密测量科学与技术创新研究院 GABA (gamma-aminobutyric acid) energy neuron specific promoter and application thereof
CN112043710A (en) * 2020-09-28 2020-12-08 广州智睿医药科技有限公司 Medicine for treating or preventing diseases related to LRRK2 kinase or abnormal LRRK2 mutant kinase activity

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
SABRINA ZECHEL ET AL.: "Expression of leucine-rich-repeat-kinase 2 (LRRK2) during embryonic development", 《INTERNATIONAL JOURNAL OF DEVELOPMENTAL NEUROSCIENCE》 *
SABRINA ZECHEL ET AL.: "Expression of leucine-rich-repeat-kinase 2 (LRRK2) during embryonic development", 《INTERNATIONAL JOURNAL OF DEVELOPMENTAL NEUROSCIENCE》, vol. 28, no. 5, 31 August 2010 (2010-08-31), pages 392 *
SCHERER,S.E. ET AL.: "NCBI Reference Sequence: NC_000012.12", 《GENBANK》 *
SCHERER,S.E. ET AL.: "NCBI Reference Sequence: NC_000012.12", 《GENBANK》, 9 December 2019 (2019-12-09) *
杨旭等: "α-突触核蛋白过表达转基因帕金森病动物模型研究进展", 《国际神经病学神经外科学杂志》 *
杨旭等: "α-突触核蛋白过表达转基因帕金森病动物模型研究进展", 《国际神经病学神经外科学杂志》, no. 03, 28 June 2012 (2012-06-28), pages 278 - 281 *
莫晓云 等: "LRRK2基因核心启动子的预测", 广西医学, vol. 35, no. 01, pages 38 - 41 *

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