Ordering
| Item | Catalog # | Description | Quantity | Price (USD) | ||
|---|---|---|---|---|---|---|
| Plasmid | 44361 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $75 | Add to Cart | |
| AAV1 | 44361-AAV1 | Virus (100 µL at titer ≥ 7×10¹² vg/mL)and Plasmid.More Information | Add to Cart | |||
| AAV2 | 44361-AAV2 | Limited Stock Available, 1 unit leftVirus (100 µL at titer ≥6×10¹² vg/mL)and Plasmid.More Information | Add to Cart | |||
| AAV5 | 44361-AAV5 | Virus (100 µL at titer ≥ 7×10¹² vg/mL)and Plasmid.More Information | Add to Cart | |||
| AAV8 | 44361-AAV8 | Virus (100 µL at titer ≥ 4×10¹² vg/mL)and Plasmid.More Information | Add to Cart | |||
| AAV9 | 44361-AAV9 | Virus (100 µL at titer ≥ 1×10¹³ vg/mL)and Plasmid.More Information | Add to Cart | |||
| AAV Retrograde | 44361-AAVrg | Virus (100 µL at titer ≥ 7×10¹² vg/mL)and Plasmid.More Information | Add to Cart | |||
| AAV PHP.eB | 44361-PHPeB | Virus (100 µL at titer ≥ 1×10¹³ vg/mL)and Plasmid.More Information | Add to Cart | |||
This material is available to academics and nonprofits only.
Backbone
- Vector backbonepAAV(Search Vector Database)
- Backbone sizew/o insert(bp)4824
- Total vector size (bp)7326
- Vector typeAAV ; Adeno Associated Viral Vector
Growth in Bacteria
- Bacterial Resistance(s)Ampicillin
- Growth Temperature37°C
- Growth Strain(s)NEB Stable
- Copy numberLow Copy
Gene/Insert
- Gene/Insert namehM3D(Gq)-mCherry
- SpeciesH. sapiens (human)
- Insert Size (bp)2502
- Promoterhuman Synapsin 1
- Tag/ Fusion Protein
- mCherry (C terminal on insert)
Cloning Information
- Cloning methodRestriction Enzyme
- 5′ cloning siteAsc I(not destroyed)
- 3′ cloning siteNhe I(not destroyed)
- 5′ sequencing primertcgtgtcgtgcctgagagcg
- 3′ sequencing primerGCATTAAAGCAGCGTATCCACATAGC (Common Sequencing Primers)
Resource Information
- Terms and Licenses
- UBMTA
- genOway Notice of RIghts
- Takara Bio Limited Use Label License (formerly Clontech)
- Industry Terms
- Not Available to Industry
- Articles Citing this Plasmid
- 44 References
Information for AAV1 (Catalog # 44361-AAV1)(Back to top)
Purpose
Ready-to-use AAV1 particles produced from pAAV-hSyn-DIO-hM3D(Gq)-mCherry (#44361). In addition to the viral particles, you will also receive purified pAAV-hSyn-DIO-hM3D(Gq)-mCherry plasmid DNA.
Syn-driven, Cre-dependent, hM3D(Gq) receptor with an mCherry reporter for CNO-induced neuronal activation. These AAV preparations are suitable purity for injection into animals.Delivery
- Volume100 µL
- Titer≥ 7×10¹² vg/mL
- Pricing$350 USD for preparation of 100 µL virus + $30 USD for plasmid.
- StorageStore at -80℃. Thaw just before use and keep on ice.
- ShipmentViral particles are shipped frozen on dry ice. Plasmid DNA (≥ 200ng) will also be included in the shipment.
Viral Production & Use
- Packaging Plasmidsencode adenoviral helper sequences and AAV rep gene, AAV1 cap gene
- BufferPBS + 0.001% Pluronic F-68 + 200 mM NaCl
- SerotypeAAV1
- PurificationIodixanol gradient ultracentrifugation
- Reporter GenemCherry (Cre-dependent)
Biosafety
Requestor is responsible for compliance withtheir institution"s biosafety regulations.Lentivirus is generally considered BSL-2. AAV isgenerally considered BSL-1, but may requireBSL-2 handling depending on the insert.Biosafety Guide
Resource Information
- Terms and Licenses
- Ancillary Agreement for Penn Vectors
- Terms of Use for Viral Vectors
- Industry Terms
- Not Available to Industry
Viral Quality Control
- Addgene ensures high quality viral vectors by optimizing and standardizing production protocols and performing rigorous quality control (QC) (see a list of our QC assays). Thespecific QC assays performed varies for each viral lot. To learn which specific QC assays were performed on your lot, please contact us.
- Titer: the exact titer of your sample will be reported on the tube. The titer you see listed on this page is the guaranteed minimum titer. See how titers are measured.
Visit our viral production page for moreinformation.
Addgene Comments
Using FLEX vectors in vivo: LoxP sites in FLEX plasmids are known to recombine during DNA amplification and viral vector production, which may result in a minority of Cre-activated (i.e., "flipped") viral vectors. Addgene has measured this occurs in 0.01-0.03% of viral vectors in our typical production protocol. This can lead to a small number of cells exhibiting Cre-independent transgene expression in vivo. To address this, we recommend titrating to find the optimal AAV dosage required for Cre-dependent transgene expression and function in vivo. This may include reducing the viral vector dosage in order to reduce the likelihood of Cre-independent expression.
Information for AAV2 (Catalog # 44361-AAV2)(Back to top)
Purpose
Ready-to-use AAV2 particles produced from pAAV-hSyn-DIO-hM3D(Gq)-mCherry (#44361). In addition to the viral particles, you will also receive purified pAAV-hSyn-DIO-hM3D(Gq)-mCherry plasmid DNA.
Syn-driven, Cre-dependent, hM3D(Gq) receptor with an mCherry reporter for CNO-induced neuronal activation. These AAV preparations are suitable purity for injection into animals.Delivery
- Volume100 µL
- Titer≥ 6×10¹² vg/mL
- Pricing$350 USD for preparation of 100 µL virus + $30 USD for plasmid.
- StorageStore at -80℃. Thaw just before use and keep on ice.
- ShipmentViral particles are shipped frozen on dry ice. Plasmid DNA (≥ 200ng) will also be included in the shipment.
Viral Production & Use
- Packaging Plasmidsencode adenoviral helper sequences and AAV rep gene, AAV2 cap gene
- BufferPBS + 0.001% Pluronic F-68
- SerotypeAAV2
- PurificationIodixanol gradient ultracentrifugation
- Reporter GenemCherry (Cre-dependent)
Biosafety
Requestor is responsible for compliance withtheir institution"s biosafety regulations.Lentivirus is generally considered BSL-2. AAV isgenerally considered BSL-1, but may requireBSL-2 handling depending on the insert.Biosafety Guide
Resource Information
- Terms and Licenses
- Terms of Use for Viral Vectors
- Industry Terms
- Not Available to Industry
Viral Quality Control
- Addgene ensures high quality viral vectors by optimizing and standardizing production protocols and performing rigorous quality control (QC) (see a list of our QC assays). Thespecific QC assays performed varies for each viral lot. To learn which specific QC assays were performed on your lot, please contact us.
- Titer: the exact titer of your sample will be reported on the tube. The titer you see listed on this page is the guaranteed minimum titer. See how titers are measured.
Visit our viral production page for moreinformation.
Addgene Comments
Using FLEX vectors in vivo: LoxP sites in FLEX plasmids are known to recombine during DNA amplification and viral vector production, which may result in a minority of Cre-activated (i.e., "flipped") viral vectors. Addgene has measured this occurs in 0.01-0.03% of viral vectors in our typical production protocol. This can lead to a small number of cells exhibiting Cre-independent transgene expression in vivo. To address this, we recommend titrating to find the optimal AAV dosage required for Cre-dependent transgene expression and function in vivo. This may include reducing the viral vector dosage in order to reduce the likelihood of Cre-independent expression.
Data submitted about 44361-AAV2 by requesting scientist(s):
- Data 1: Mouse, Retina
- Data 2: Rat, Brain parenchyma
Information for AAV5 (Catalog # 44361-AAV5)(Back to top)
Purpose
Ready-to-use AAV5 particles produced from pAAV-hSyn-DIO-hM3D(Gq)-mCherry (#44361). In addition to the viral particles, you will also receive purified pAAV-hSyn-DIO-hM3D(Gq)-mCherry plasmid DNA.
Syn-driven, Cre-dependent, hM3D(Gq) receptor with an mCherry reporter for CNO-induced neuronal activation. These AAV preparations are suitable purity for injection into animals.Delivery
- Volume100 µL
- Titer≥ 7×10¹² vg/mL
- Pricing$350 USD for preparation of 100 µL virus + $30 USD for plasmid.
- StorageStore at -80℃. Thaw just before use and keep on ice.
- ShipmentViral particles are shipped frozen on dry ice. Plasmid DNA (≥ 200ng) will also be included in the shipment.
Viral Production & Use
- Packaging Plasmidsencode adenoviral helper sequences and AAV rep gene, AAV5 cap gene
- BufferPBS + 0.001% Pluronic F-68
- SerotypeAAV5
- PurificationIodixanol gradient ultracentrifugation
- Reporter GenemCherry (Cre-dependent)
Biosafety
Requestor is responsible for compliance withtheir institution"s biosafety regulations.Lentivirus is generally considered BSL-2. AAV isgenerally considered BSL-1, but may requireBSL-2 handling depending on the insert.Biosafety Guide
Resource Information
- Terms and Licenses
- Terms of Use for Viral Vectors
- Industry Terms
- Not Available to Industry
Viral Quality Control
- Addgene ensures high quality viral vectors by optimizing and standardizing production protocols and performing rigorous quality control (QC) (see a list of our QC assays). Thespecific QC assays performed varies for each viral lot. To learn which specific QC assays were performed on your lot, please contact us.
- Titer: the exact titer of your sample will be reported on the tube. The titer you see listed on this page is the guaranteed minimum titer. See how titers are measured.
Visit our viral production page for moreinformation.
Addgene Comments
Using FLEX vectors in vivo: LoxP sites in FLEX plasmids are known to recombine during DNA amplification and viral vector production, which may result in a minority of Cre-activated (i.e., "flipped") viral vectors. Addgene has measured this occurs in 0.01-0.03% of viral vectors in our typical production protocol. This can lead to a small number of cells exhibiting Cre-independent transgene expression in vivo. To address this, we recommend titrating to find the optimal AAV dosage required for Cre-dependent transgene expression and function in vivo. This may include reducing the viral vector dosage in order to reduce the likelihood of Cre-independent expression.
Data submitted about 44361-AAV5 by requesting scientist(s):
- Data 1: Mouse, Brain parenchyma
Information for AAV8 (Catalog # 44361-AAV8)(Back to top)
Purpose
Ready-to-use AAV8 particles produced from pAAV-hSyn-DIO-hM3D(Gq)-mCherry (#44361). In addition to the viral particles, you will also receive purified pAAV-hSyn-DIO-hM3D(Gq)-mCherry plasmid DNA.
Syn-driven, Cre-dependent, hM3D(Gq) receptor with an mCherry reporter for CNO-induced neuronal activation. These AAV preparations are suitable purity for injection into animals.Delivery
- Volume100 µL
- Titer≥ 4×10¹² vg/mL
- Pricing$350 USD for preparation of 100 µL virus + $30 USD for plasmid.
- StorageStore at -80℃. Thaw just before use and keep on ice.
- ShipmentViral particles are shipped frozen on dry ice. Plasmid DNA (≥ 200ng) will also be included in the shipment.
Viral Production & Use
- Packaging Plasmidsencode adenoviral helper sequences and AAV rep gene, AAV8 cap gene
- EnvelopeAAV8 cap gene
- BufferPBS + 0.001% Pluronic F-68
- SerotypeAAV8
- PurificationIodixanol gradient ultracentrifugation
- Reporter GenemCherry (Cre-dependent)
Biosafety
Requestor is responsible for compliance withtheir institution"s biosafety regulations.Lentivirus is generally considered BSL-2. AAV isgenerally considered BSL-1, but may requireBSL-2 handling depending on the insert.Biosafety Guide
Resource Information
- Terms and Licenses
- Ancillary Agreement for Penn Vectors
- Terms of Use for Viral Vectors
- Industry Terms
- Not Available to Industry
Viral Quality Control
- Addgene ensures high quality viral vectors by optimizing and standardizing production protocols and performing rigorous quality control (QC) (see a list of our QC assays). Thespecific QC assays performed varies for each viral lot. To learn which specific QC assays were performed on your lot, please contact us.
- Titer: the exact titer of your sample will be reported on the tube. The titer you see listed on this page is the guaranteed minimum titer. See how titers are measured.
Visit our viral production page for moreinformation.
Addgene Comments
Using FLEX vectors in vivo: LoxP sites in FLEX plasmids are known to recombine during DNA amplification and viral vector production, which may result in a minority of Cre-activated (i.e., "flipped") viral vectors. Addgene has measured this occurs in 0.01-0.03% of viral vectors in our typical production protocol. This can lead to a small number of cells exhibiting Cre-independent transgene expression in vivo. To address this, we recommend titrating to find the optimal AAV dosage required for Cre-dependent transgene expression and function in vivo. This may include reducing the viral vector dosage in order to reduce the likelihood of Cre-independent expression.
Data submitted about 44361-AAV8 by requesting scientist(s):
- Data 1: Mouse, Brain parenchyma
- Data 2: Mouse, Brain parenchyma
- Data 3: Mouse, Brain parenchyma
- Data 4: Mouse, Brain parenchyma
- Data 5: Rat, Brain parenchyma
- Data 6: Mouse, Brain parenchyma
Information for AAV9 (Catalog # 44361-AAV9)(Back to top)
Purpose
Ready-to-use AAV9 particles produced from pAAV-hSyn-DIO-hM3D(Gq)-mCherry (#44361). In addition to the viral particles, you will also receive purified pAAV-hSyn-DIO-hM3D(Gq)-mCherry plasmid DNA.
Syn-driven, Cre-dependent, hM3D(Gq) receptor with an mCherry reporter for CNO-induced neuronal activation. These AAV preparations are suitable purity for injection into animals.Delivery
- Volume100 µL
- Titer≥ 1×10¹³ vg/mL
- Pricing$350 USD for preparation of 100 µL virus + $30 USD for plasmid.
- StorageStore at -80℃. Thaw just before use and keep on ice.
- ShipmentViral particles are shipped frozen on dry ice. Plasmid DNA (≥ 200ng) will also be included in the shipment.
Viral Production & Use
- Packaging Plasmidsencode adenoviral helper sequences and AAV rep gene, AAV9 cap gene
- BufferPBS + 0.001% Pluronic F-68
- SerotypeAAV9
- PurificationIodixanol gradient ultracentrifugation
- Reporter GenemCherry (Cre-dependent)
Biosafety
Requestor is responsible for compliance withtheir institution"s biosafety regulations.Lentivirus is generally considered BSL-2. AAV isgenerally considered BSL-1, but may requireBSL-2 handling depending on the insert.Biosafety Guide
Resource Information
- Terms and Licenses
- Ancillary Agreement for Penn Vectors
- Terms of Use for Viral Vectors
- Industry Terms
- Not Available to Industry
Viral Quality Control
- Addgene ensures high quality viral vectors by optimizing and standardizing production protocols and performing rigorous quality control (QC) (see a list of our QC assays). Thespecific QC assays performed varies for each viral lot. To learn which specific QC assays were performed on your lot, please contact us.
- Titer: the exact titer of your sample will be reported on the tube. The titer you see listed on this page is the guaranteed minimum titer. See how titers are measured.
Visit our viral production page for moreinformation.
Addgene Comments
Using FLEX vectors in vivo: LoxP sites in FLEX plasmids are known to recombine during DNA amplification and viral vector production, which may result in a minority of Cre-activated (i.e., "flipped") viral vectors. Addgene has measured this occurs in 0.01-0.03% of viral vectors in our typical production protocol. This can lead to a small number of cells exhibiting Cre-independent transgene expression in vivo. To address this, we recommend titrating to find the optimal AAV dosage required for Cre-dependent transgene expression and function in vivo. This may include reducing the viral vector dosage in order to reduce the likelihood of Cre-independent expression.
Information for AAV Retrograde (Catalog # 44361-AAVrg)(Back to top)
Purpose
Ready-to-use AAV Retrograde particles produced from pAAV-hSyn-DIO-hM3D(Gq)-mCherry (#44361). In addition to the viral particles, you will also receive purified pAAV-hSyn-DIO-hM3D(Gq)-mCherry plasmid DNA.
Syn-driven, Cre-dependent, hM3D(Gq) receptor with an mCherry reporter for CNO-induced neuronal activation. These AAV were produced with a retrograde serotype, which permits retrograde access to projection neurons. These AAV preparations are suitable purity for injection into animals.Delivery
- Volume100 µL
- Titer≥ 7×10¹² vg/mL
- Pricing$350 USD for preparation of 100 µL virus + $30 USD for plasmid.
- StorageStore at -80℃. Thaw just before use and keep on ice.
- ShipmentViral particles are shipped frozen on dry ice. Plasmid DNA (≥ 200ng) will also be included in the shipment.
Viral Production & Use
- Packaging Plasmidsencode adenoviral helper sequences and AAV rep gene, AAV retrograde cap gene from rAAV2-retro helper (plasmid #81070)
- BufferPBS + 0.001% Pluronic F-68 + 200 mM NaCl
- SerotypeAAV retrograde (AAVrg)
- PurificationIodixanol gradient ultracentrifugation
- Reporter GenemCherry (Cre-dependent)
Biosafety
Requestor is responsible for compliance withtheir institution"s biosafety regulations.Lentivirus is generally considered BSL-2. AAV isgenerally considered BSL-1, but may requireBSL-2 handling depending on the insert.Biosafety Guide
Resource Information
- Terms and Licenses
- Terms of Use for Viral Vectors
- Industry Terms
- Not Available to Industry
Viral Quality Control
- Addgene ensures high quality viral vectors by optimizing and standardizing production protocols and performing rigorous quality control (QC) (see a list of our QC assays). Thespecific QC assays performed varies for each viral lot. To learn which specific QC assays were performed on your lot, please contact us.
- Titer: the exact titer of your sample will be reported on the tube. The titer you see listed on this page is the guaranteed minimum titer. See how titers are measured.
Visit our viral production page for moreinformation.
Addgene Comments
Retrograde functionality is dependent on high viral titers. Addgene recommends not diluting your AAV preps prior to use.Using FLEX vectors in vivo: LoxP sites in FLEX plasmids are known to recombine during DNA amplification and viral vector production, which may result in a minority of Cre-activated (i.e., "flipped") viral vectors. Addgene has measured this occurs in 0.01-0.03% of viral vectors in our typical production protocol. This can lead to a small number of cells exhibiting Cre-independent transgene expression in vivo. To address this, we recommend titrating to find the optimal AAV dosage required for Cre-dependent transgene expression and function in vivo. This may include reducing the viral vector dosage in order to reduce the likelihood of Cre-independent expression.
Data submitted about 44361-AAVrg by requesting scientist(s):
- Data 1: Mouse, Brain parenchyma
Information for AAV PHP.eB (Catalog # 44361-PHPeB)(Back to top)
Purpose
Ready-to-use AAV PHP.eB particles produced from pAAV-hSyn-DIO-hM3D(Gq)-mCherry (#44361). In addition to the viral particles, you will also receive purified pAAV-hSyn-DIO-hM3D(Gq)-mCherry plasmid DNA.
Syn-driven, Cre-dependent, hM3D(Gq) receptor with an mCherry reporter for CNO-induced neuronal activation. These AAV were produced with the PHP.eB serotype, which permits efficient transduction of the central nervous system.These AAV preparations are suitable purity for injection into animals.Delivery
- Volume100 µL
- Titer≥ 1×10¹³ vg/mL
- Pricing$350 USD for preparation of 100 µL virus + $30 USD for plasmid.
- StorageStore at -80℃. Thaw just before use and keep on ice.
- ShipmentViral particles are shipped frozen on dry ice. Plasmid DNA (≥ 200ng) will also be included in the shipment.
Viral Production & Use
- Packaging Plasmidsencode adenoviral helper sequences and AAV rep gene, PHP.eB cap gene pUCmini-iCAP-PHP.eB (plasmid #103005)
- BufferPBS + 0.001% Pluronic F-68
- SerotypePHPeB (plasmid #103005)
- PurificationIodixanol gradient ultracentrifugation
- Reporter GenemCherry (Cre-dependent)
Biosafety
Requestor is responsible for compliance withtheir institution"s biosafety regulations.Lentivirus is generally considered BSL-2. AAV isgenerally considered BSL-1, but may requireBSL-2 handling depending on the insert.Biosafety Guide
Resource Information
- Terms and Licenses
- Ancillary Agreement for Penn Vectors
- Terms of Use for Viral Vectors
- Industry Terms
- Not Available to Industry
Viral Quality Control
- Addgene ensures high quality viral vectors by optimizing and standardizing production protocols and performing rigorous quality control (QC) (see a list of our QC assays). Thespecific QC assays performed varies for each viral lot. To learn which specific QC assays were performed on your lot, please contact us.
- Titer: the exact titer of your sample will be reported on the tube. The titer you see listed on this page is the guaranteed minimum titer. See how titers are measured.
Visit our viral production page for moreinformation.
Addgene Comments
Using FLEX vectors in vivo: LoxP sites in FLEX plasmids are known to recombine during DNA amplification and viral vector production, which may result in a minority of Cre-activated (i.e., "flipped") viral vectors. Addgene has measured this occurs in 0.01-0.03% of viral vectors in our typical production protocol. This can lead to a small number of cells exhibiting Cre-independent transgene expression in vivo. To address this, we recommend titrating to find the optimal AAV dosage required for Cre-dependent transgene expression and function in vivo. This may include reducing the viral vector dosage in order to reduce the likelihood of Cre-independent expression.
Citation Information: When using the PHP.eB serotype in future publications, please acknowledge Viviana Gradinaru and cite Chan et al., Nat Neurosci, 20(8):1172-1179. Pubmed.ebiomall.com
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客户提供：
1.目的基因信息(GenBankAccessionnumber、GeneID、或关键词)。
2.细胞系和培养条件
缺点：质粒仍然较大，转染难度相对较大。具有碱基识别偏好性，局限了基因编辑的运用范围，而且会导致不同基因位点编辑效率不同。筛选仍然需要较大工作量。
http://www.nature.com/nature/journal/v520/n7546/full/nature14299.html
请教各位大神，CRISPR/Cas9基因敲除的小鼠（完全性敲除）构建后，鉴定的方法是不是只要PCR扩增进行基因型鉴定就可以了？核型鉴定及southernblot检测需不需要呢？谢谢！
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近年来，CRISPR/Cas9风暴席卷全球。这项基因组编辑技术在短短几年内迅速应用于世界各地的实验室，并催生了上千篇文章的发表。然而，麻省理工学院（MIT）的《Technology Review》杂志提出了一个问题，谁才是CRISPR技术的所有者？生物通 www.ebiotrade.com
一个月前，“科学突破奖”（Breakthrough Prize）揭晓。加州大学伯克利分校的Jennifer Doudna和德国亥姆霍兹传染研究中心的Emmanuelle Charpentier因在CRISPR技术方面的重要贡献而获奖，且每人获得了300万美元的奖金。
这一领域的另一位风云人物，Broad研究院的张锋（Feng Zhang）虽然没有获得科学突破奖，但在今年4月获得了CRISPR/Cas9的首个专利。他的研究中心控制着这一技术的每个重要商业应用。生物通 www.ebiotrade.com
那么问题来了，这个备受瞩目的奖项和专利为何会落入不同人的手中？究竟是谁发明了它？“这一领域的知识产权相当复杂，”CRISPR Therapeutics公司的CEO Rodger Novak谈道。Emmanuelle Charpentier是这家公司的创始人之一。
Tech Review的记者Antonio Regalado指出，张锋与Doudna共同创立了Editas Medicine公司，它获得了Broad的技术授权。不过，在张锋成功申请专利之后，Doudna就与该公司断绝了关系。她将她的知识产权（专利申请中）授予了另一家竞争性的公司Intellia Therapeutics。然而，让事情更加复杂的是，Charpentier又将她在同一专利申请中的权利出售给了CRISPR Therapeutics。生物通 www.ebiotrade.com
此外，围绕这一技术的科学信用，那也是错综复杂。2012年夏天，Doudna、Charpentier及她们的团队发表了一篇文章，证明CRISPR/Cas9系统可以作为一种可编程的DNA编辑工具。半年后，张锋博士以及哈佛大学的George Church发表文章称它可应用于人类细胞，不久后，Doudna也发表了类似的结果。
但是，张锋表示他对Doudna和Charpentier的工作知之甚少。为了支持他的专利申请，他提交了实验室笔记本的照片，表示他在2012年年初就开始了这方面的研究，早于Doudna和Charpentier。生物通 www.ebiotrade.com
Charpentier表示：“我可以说的是，我和Jennifer Doudna是在我的实验室中开展研究。这里的一切都很夸张，因为这是一项人们很容易学会的技术。事情发生得太快了。”
Regalado在文中写道：“目前还没有CRISPR药物。但是如果CRISPR真的如科学家想象得那么重要，对这一基本技术的商业控制将价值数十亿美元。”也许，这场专利之战才刚刚开始。（生物通 薄荷）
基因敲除、阳性单克隆筛选、真核细胞、斑马鱼、基因组、植物
CRISPR（ClusteredRegularlyInterspacedShortPalindromicRepeats）RNA，是最近几年才发现的原核生物中的调控RNA，用以抵御病毒和质粒入侵。在II型CRISPR系统中，CRISPRRNA（crRNA）与转录激活crRNA（Trans-activatingcrRNA,tracrRNA）退火形成的复合物能特异识别基因组序列，引导Cas9核酸内切酶在目的片段生成DNA双链断裂（double-strandbreaks,DSBs）。CRISPR-Cas系统的高效基因组编辑功能已被应用于多种生物，包括人、小鼠、大鼠、斑马鱼、秀丽隐杆线虫、植物及细菌。多个科研小组的研究都显示，与锌指核酸酶（ZFNs）和转录激活样效应核酸酶（Transcriptionactivator-likeeffectornucleases,TALEN）相比较，CRISPR-Cas系统介导的基因组靶向实验在真核细胞中具有相似甚至更高的效率。
剪刀手生物专注于基因编辑多年，最新推出基因组编辑工具CRISPR/Cas9专家系统，该系统灵活简单、可以对特定基因组位点进行切割置换，特异性高、细胞毒性低。CRISPR/Cas9系统可广泛应用于基因组工程，如基因抑制，基因敲除，基因敲入，基因修复等。CRISPR-Cas9体系为基因组工程研究提供了一项简便而强大的工具。
作为一种新的基因编辑技术，CRISPR/Cas9有以下特点和优势：
1、操作简单，靶向精确性更高。
2、CRISPR/Cas9系统是由RNA调控的对DNA的修饰，其基因修饰可遗传。
3、基因修饰率高，基因调控方式多样，例如敲除、插入、抑制、激活等
4、可实现对靶基因多个位点同时敲除。
5、无物种限制。
6、实验周期短，最快仅需1个月，节省大量时间和成本。
CRISPR/Cas9的应用中，活性检测或是突变效率的检测：
错配酶法靶序列经Cas/sgRNA切割后由于缺乏修复模板，将主要以非同源重组的方式进行修复，或多或少会插入或删除一些碱基。因此将靶序列PCR扩增后经变性、退火，将形成错配。错配酶将识别错配的杂合双链并剪切。产物跑电泳，比较切割条带与未切割条带的比例，即可反映出Cas/sgRNA的活性。
http://www.addgene.org/crispr/guide/
一、Cas9简介
二、如何设计gRNA
三、如何快速克隆gRNA
四、如何制作基因敲除、基因敲入和条件性敲除
A
B
C条件性敲除
目前的条件性敲除小鼠主要是基于Cre-LoxP系统的。Cre-LoxP系统是源于P1噬菌体的一个DNA重组体系,由Cre酶和相应的LoxP位点组成,它能导致重组发生在特定的DNA序列处(LoxP位点),该系统可以将外源基因定点整合到染色体上或将特定DNA片段删除。
传统的条件性敲除
基于Cre-LoxP的基因打靶要分两步来进行。
传统方法，首先要在胚胎干细胞的基因组中引入LoxP序列，这一步可以通过打靶载体的设计和对同源重组子的筛选来实现。
第二步，通过Cre介导的重组来实现靶基因的遗传修饰或改变。在细胞水平上，可以用Cre重组酶表达质粒转染中靶细胞，通过识别LoxP位点将抗性标记基因切除；在个体水平上将重组杂合子小鼠与Cre转基因小鼠杂交，筛选子代小鼠就可得到删除外源标记基因的条件性敲除小鼠。将Cre基因置于可诱导的启动子控制下，通过诱导表达Cre重组酶而将LoxP位点之间的基因切除（诱导性基因敲除），实现特定基因在特定时间或者组织中的失活。
一般需要一年以上的时间，价格在15-20万不等。
基于Cas9系统的条件性敲除
原理上和传统的条件性敲除类似，差别在于利用OptimizedCas9/CRISPRSystem(OCAS)技术在基因组上加入loxp序列，可以快速获得Loxp位点插入的小鼠。
一般只需要4-5个月。价格也比传统方法大大降低。
五、如何制作基因敲除小鼠
因为技术和仪器的缺陷，一般实验室都不太会做到这一步。
mark先定格框架慢慢写
还开了另外一个帖子讲Cas9技术本身及在细胞生物学上的应用。也欢迎交流。
Cas9基因敲除技术详述-蚂蚁淘论坛
