shRNA遺伝子KDソリューションズ

VectorBuilder offers a comprehensive collection of shRNA reagents to provide you with the ideal tools for your RNAi experiments. We offer both U6 and miR30-based shRNA systems for providing you with the flexibility to control shRNA expression in different ways based on your experimental needs. We can package all major virus types (e.g. lentivirus, AAV, adenovirus) at various titer scales for delivering your shRNAs into difficult-to-transfect cells. We are also specialized in building pooled shRNA libraries for large-scale loss-of-function screens in mammalian cells. Additionally, our online vector design platform is integrated with shRNA databases for popular species, enabling you to easily select suitable shRNAs to target your gene of interest (GOI).
特徴
  • Highly intuitive online design platform with whole-genome shRNA databases for quick and easy designing of shRNA vectors
  • Rich collections of vector backbones and vector components
  • Premade and custom-made shRNA libraries available
  • 100% sequence validated, fast turnaround and competitive pricing
  • Powerful technical support for shRNA selection, vector design and troubleshooting
受託サービスの種類
  • カスタムshRNAベクター
  • ポピュラーshRNAベクター
  • shRNAウイルス
  • shRNA (3+1)ウイルスパッケージング
  • プール型shRNAライブラリー
  • shRNAノックダウン安定細胞株
テクニカルインフォメーション
  • shRNAを介した遺伝子ノックダウン
  • shRNAの発現コントロール 
  • shRNAデータベース

受託サービスの種類

カスタムshRNAベクター

Using our high-intuitive online vector design platform, you can choose from over 30 vector backbones (non-viral, viral or transposon) and unlimited combinations of vector components (promoters, fluorescent and drug-selection markers) for expressing your shRNA. Our shRNA databases allow you to easily select shRNAs against your GOI, without the need to design them yourself. We also offer shRNA sensor vectors for testing the knockdown efficiency of the shRNA of interest.

Plasmid DNA preparation and virus packaging can be purchased as downstream services upon finishing the designs of shRNA vectors.

カスタムshRNAベクターをデザインする 開く

In addition to the vector systems listed above, we can design and construct IPTG-inducible shRNA expression vectors and Cre-lox based conditional shRNA expression vectors. Just デザインリクエストを送る!

ベクター構築サービスについて
ポピュラーshRNAベクター

VectorBuilder offers a panel of popular shRNA vectors that express either scramble shRNA or shRNAs targeting popular genes suitable for being used as control vectors in many biological applications. Plasmid DNA preparation and virus packaging can be purchased as downstream services upon adding these vectors into shopping cart.

You can use the vector picker below for ordering U6-based popular shRNA vectors.

For ordering miR30-based popular shRNA vectors, simply デザインリクエストを送る describing your needs.

shRNAウイルス

VectorBuilder offers premium quality virus packaging services for lentivirus, AAV and adenovirus in a variety of scales for delivering shRNA into difficult-to-transfect cells. Our proprietary technologies and reagents have greatly improved virus packaging protocols in terms of titer, purity, viability and consistency. Our packaging protocols are also optimized for the viral vector systems used in our vector construction services. As a result, we have a growing base of highly satisfied customers who come back to us again and again for their cloning and virus packaging needs.

レンチウイルスパッケージングサービスの注文情報 開く
サイズ 使用目的 標準タイター ミニマムタイター 容量 価格(税抜き) 作業日数
パイロット 培養細胞 >4x10TU/ml >10TU/ml 250 ul (10x25 ul) 66,000円 8-16 営業日
中容量 >3x10TU/ml 1 ml (10x100 ul) 92,000円
大容量 >2x10TU/ml >10TU/ml 1 ml (10x100 ul) 144,000円
超純粋中容量

培養細胞& in vivo

>2x10TU/ml >10TU/ml 500 ul (10x50 ul) 170,000円
超純粋大容量 1 ml (10x 100 ul) 209,000円

TU = Transduction units (also known as infectious units)

AAVパッケージングサービスの注文情報  開く
サイズ 推奨使用系 標準タイター ミニマムタイター 容量 価格(税抜き) 作業日数
パイロット 培養細胞 >1012 GC/ml >2x1011 GC/ml 250 ul (10x25 ul) 66,000円 10-20営業日
中容量 1 ml (10x100 ul) 85,500円
大容量 >5x1012 GC/ml >2x1012 GC/ml 1 ml (10x100 ul) 144,000円
超純粋パイロット 培養細胞 & in vivo >2x1013 GC/ml >1013 GC/ml 100 ul (4x25 ul) 163,500円 20-30営業日
超純粋中容量 500 ul (10x50 ul) 229,000円
超純粋大容量 1 ml (10x100 ul) 345,500円

GC = Genome copies

アデノウイルスパッケージングサービスの注文情報  開く
サイズ 推奨使用系 標準タイター ミニマムタイター 容量 価格(税抜き) 作業日数
パイロット 培養細胞 >2x1010 PFU/ml >1010 PFU/ml 250 ul (10x25 ul) 66,000円 20-32 営業日
中容量 1 ml (10x100 ul) 92,000円
大容量 >2x1011 PFU/ml >1011 PFU/ml 1 ml (10x100 ul) 144,000円
超純粋大容量 培養細胞 & in vivo >2x1012 VP/ml >1012 VP/ml 1 ml (10x100 ul) 209,000円 22-36 営業日

PFU = Plaque forming units; VP = Virus particles

ウイルスパッケージングサービスについて

shRNA (3+1)ウイルスパッケージング

It is important to recognize the fact that not all empirically designed shRNAs will work. The potency of an shRNA is determined by several factors including the length of the shRNA, loop structure, GC profile and thermodynamic stability of the shRNA, secondary structure of the target sequence, and off-target matches to other genes. Typically, ~50-70% of shRNAs have a noticeable knockdown effect, and ~20-30% of them have a strong knockdown. Therefore, it is important to test multiple shRNAs to find the most potent one for downstream experiments.

VectorBuilder offers shRNA (3+1) virus packaging services which include 3 custom shRNA viruses targeting your GOI and 1 scramble control virus, enabling you to test multiple shRNAs against your target gene at highly affordable prices. This service is currently available for lentivirus, AAV and adenovirus.

shRNA (3+1) ウイルスパッケージングサービスの注文情報 開く
ウイルス種 サイズと納品形態  推奨使用系 価格 (税抜き)* 作業日数**
レンチウイルス パイロット 培養細胞 176,500円 15-30 営業日
中容量 241,500円
大容量 371,500円
超純粋中容量 培養細胞 & in vivo 488,500円
超純粋大容量 592,500円
AAV パイロット 培養細胞’ 176,500円 17-34 営業日
中容量 241,500円
大容量 371,500円
超純粋パイロット 培養細胞 & in vivo 488,500円 27-44 営業日
超純粋中容量 657,500円
超純粋大容量’ 982,500円
アデノウイルス パイロット 培養細胞’ 222,000円 35-58 営業日
中容量 287,000円
大容量 471,000円
超純粋大容量 培養細胞 & in vivo 638,000円 37-62 営業日

* 価格には、ベクター構築とウイルスパッケージングの全てを含みます。

** 作業日数はベクター構築とウイルスパッケージングの両工程を合計しています。

下の選択ツールを使って目的遺伝子のshRNA (3+1) ウイルスパッケージングを注文する

Please note that the vector picker above applies for U6-based shRNA vectors only. If you need miR30-based shRNA vectors or if you are not able to find desired shRNAs for your target gene using the vector picker, simply send us a design request describing your needs.

shRNAライプラリー(プール型)

Pooled shRNA libraries can serve as powerful and cost-efficient tools for performing large-scale loss-of-function screens for genes involved in disease pathways, cell responses to drug treatment, developmental processes, gene regulation, etc. We can deliver your library as E. coli stock, plasmid DNA pool, or packaged virus, depending on your needs. Our custom libraries are fully validated by next generation sequencing so that you know exactly what you get.

In addition to custom pooled shRNA library construction, VectorBuilder offers high-quality premade pooled shRNA libraries targeting human and mouse genes. For each species, we provide ready-to-use lentivirus libraries at two scales: Whole Genome (~19,000 RefSeq genes) and Elite Gene (~2,000 most frequently cited genes on PubMed Central). Where possible, each gene is targeted by 5-6 different shRNAs. These libraries have been fully validated by next-generation sequencing and functional assays.

既製品shRNAライブラリーの特徴:

  • Whole-genome and high-coverage targeting
  • Validation of library quality by NGS
  • High uniformity
  • Available as ready-to-use high-titer lentivirus
  • Dual EGFP/Puro marker for efficient and versatile selection or tracking of positively transduced cells
プリメイド(既製品)shRNAライブラリーの注文情報 開く
製品名 遺伝子数 shRNA数 サイズ* カタログ番号 価格(税抜き)
ヒト エリート遺伝子 プール型 shRNA ライブラリー 2,161 12,471 中容量
(>1.0x108 TU/ml, 1 ml)
LVM(Lib190505-1037bjk)

339,000 円

639,000 円

マウス エリート遺伝子 プール型shRNA ライブラリー 2,233 12,472 中容量
(>1.0x108 TU/ml, 1 ml)
LVM(Lib190505-1039sgb)

339,000 円

639,000 円

ヒト ホールゲノム プール型shRNA ライブラリー 18,432 92,917 中容量
(>1.0x108 TU/ml, 1 ml)
LVM(Lib190505-1038ngq)

339,000 円

639,000 円

プラス
(>1.0x108 TU/ml, 5 ml)
LV5M(Lib190505-1038ngq)

989,000 円

1,889,000 円

マウス ホールゲノム プール型 shRNA ライブラリー 19,790 92,917 中容量
(>1.0x108 TU/ml, 1 ml)
LVM(Lib190505-1042tfx)

339,000 円

639,000 円

プラス
(>1.0x108 TU/ml, 5 ml)
LV5M(Lib190505-1042tfx)

989,000 円

1,889,000 円

* エリート遺伝子ライブラリー:中容量は 100 x shRNAカバレッジで、 >40スクリーニングを実施できます。ホールゲノムライブラリー:中容量は 100 x shRNAカバレッジで、 >5 スクリーニングを実施できます。プラススケールの場合、 100x shRNAカバレッジで、>25 スクリーニングを実施できます。

shRNAノックダウン安定細胞株

VectorBuilder can custom build shRNA knockdown stable cell lines for applications requiring long-term knockdown of your GOI. To ensure efficient knockdown of your GOI, the top 3 candidate shRNAs based on knockdown score are tested and the one with the best knockdown efficiency is then used for generating the stable cell line via lentivirus transduction. The knockdown level of the cell line is validated by RT-qPCR. Additionally, a series of standard QC assays such as sterility tests and mycoplasma detection are performed for releasing the final cell line products.

安定細胞株受託構築の注文情報  開く
安定細胞株モデル 樹立方法 納品形態 価格 (税抜き) 作業日数
shRNA遺伝子ノックダウン レンチウイルスでの遺伝子導入 細胞プール 878,000円より 8-13週
3種の独立した細胞クローン 1,138,000円より 11-18週

テクニカルインフォメーション

shRNAを介した遺伝子ノックダウン

Short hairpin RNA (shRNAs) are RNA molecules with stem-loop structures that can be used for targeted degradation of mRNA sequences through complementary base-pairing and therefore, are widely used for a variety of RNAi applications. shRNAs can be introduced into target cells using double-stranded DNA vectors, in both viral and non-viral formats. When cells are transfected or transduced with a shRNA vector, the shRNA is transcribed in the nucleus to form a hairpin structure consisting of a sense strand having the same sequence as the mRNA to be silenced, followed by a single-stranded loop and then an antisense strand, which is complementary to the sense strand. The transcribed shRNA exits the nucleus, is processed by Dicer in the cytoplasm and is then loaded onto the RNA-induced silencing complex (RISC) complex for subsequent target mRNA recognition and degradation (Figure 1).

図 1 . U6-ベースと miR-ベースの shRNAを介した遺伝子発現ノックダウン

shRNA-mediated knockdown of gene expression offers several advantages over gene expression knockdown using conventional synthetic small interfering RNAs (siRNAs) and therefore, has gained popularity as the preferred knockdown method for most RNAi applications.

The table below summarizes the advantages of shRNA-mediated gene knockdown over siRNA-mediated gene knockdown:

shRNAを介したノックダウン siRNAを介したノックダウン
デリバリー方法 Transfection or transduction depending on vector type Transfection
ノックダウンされる期間 Long-term Transient

エピソーマルまたは安定インテグレーション 

Can be either episomal or stable depending upon delivery method Episomal
Ability to add selection markers Yes, fluorescent or drug-selection markers can be added No
Cell-type range Suitable for a wide range of cell types Suitable for only cells with high transfection efficiency
Off-target effects Reduced off-target effects High off-target effects
Degradation rate Low High
shRNAの発現を調節するには

There are two widely used approaches to control shRNA expression on vectors: U6-based shRNA expression and miR-based shRNA expression. While U6-based shRNA vectors drive the expression of simple stem-loop shRNAs transcribed by a RNA Polymerase III promoter such as U6, miR-based shRNA vectors are used for expressing shRNAs adapted with a microRNA scaffold under an RNA Polymerase II promoter. shRNAs expressed by both U6- and miR-based vector systems are processed by similar mechanisms within the cytoplasm ultimately leading to targeted gene silencing. However, after being transcribed within the nucleus, a miR-based shRNA, unlike an U6-based shRNA, is processed in a mechanism similar to that of a primary miRNA due to the presence of endogenous miR-based sequences within its structure (Figure 1). 

The presence of RNA polymerase II promoters in miR-based shRNA vectors allows the use of tissue-specific, inducible, or variable-strength promoters, enabling a variety of experimental applications not possible with the constitutive U6 promoter. The ability of RNA polymerase II promoters to efficiently transcribe long transcripts in the miRNA-based shRNA systems provides several additional advantages relative to other knockdown vector systems. Multiple shRNAmiRs can be transcribed as a single polycistron, which is processed to form mature shRNAs within the cell. This allows knockdown of multiple genes or targeting of multiple regions within the same gene using a single transcript. As a result, this vector can be used for expressing either single or multiple shRNAmiRs. Secondly, in this vector system, a user-selected protein coding gene can be positioned within the same polycistron as the shRNAmiRs. The expression of this ORF can be used to directly monitor shRNA transcription (if a marker ORF is used) or can be used for other purposes requiring co-expression of an ORF and shRNA(s).

Despite of the flexibility of controlling shRNA expression using miR-based vectors, we often observed more robust shRNA mediated gene knockdown using U6-based vectors than miR-based vectors. Therefore, unless necessary to use miR-based shRNA vectors, in general we recommend using U6-based shRNA vectors for your gene knockdown experiments.

The table below compares a U6-based shRNA vector system and a miR-based shRNA vector system:

U6-Based shRNA Vector miR-Based shRNA Vector
shRNA structure Simple stem-loop shRNA shRNA adapted with a microRNA scaffold
shRNA length 50-70 nt >250 nt
Promoter RNA Pol III promoters such as U6 and H1 RNA Pol II promoters including ubiquitous, tissue-specific and inducible promoters
shRNA processing mechanism Processed by only Dicer in the cytoplasm Processed by Drosha in the nucleus and Dicer in the cytoplasm
No. of shRNAs that can be expressed on one vector Single shRNA (usually) Single or multiple shRNAs
Ability to express other ORFs in the shRNA transcript No Yes
Gene knockdown efficiency Often more robust Often less robust
Toxicity High cellular toxicity Decreased cellular toxicity
shRNAデータベース

VectorBuilder’s online shRNA vector design tool features optimized shRNA databases for common species, enabling you to design shRNA vectors with high knockdown efficiency for your target genes. For designing shRNAs we apply rules like those used by the RNAi consortium. All scores are ≥0, with mean at ~5, standard deviation at ~5, and 95% of scores ≤15. An shRNA with a knockdown score about 15 is considered to have the best knockdown performance and clonability, while an shRNA with a knockdown score of 0 has the worst knockdown performance or is hard to be cloned.

When you design shRNA vectors on VectorBuilder’s online platform, you will have the option to search for your target genes in our database. Upon entering your gene name, you will see detailed information on all shRNAs against your target gene available in our database, including a link to UCSC Genome Browser to view these shRNAs in the context of genomic sequence and all the transcript isoforms. Our database ranks all available shRNAs for a target gene in order of their decreasing knockdown scores and recommends testing the top 3 shRNAs with the highest knockdown scores. Please note that knockdown scores are only a rough guide. Actual knockdown efficiency could depart significantly from what the scores predict. Target sites with low scores may still work well. Also, please note that targeting 3’ UTR can be as effective as targeting coding region.

VectorBuilder’s online “Resources” contains rich educational materials to help you to successfully plan, execute and troubleshoot your shRNA-based RNAi experiments.

Click to read guides on shRNA vector systems
Click to read guides on various vector components for customizing your shRNA vectors
参考文献

Mol Cell. 9:1327 (2002); Characterization of miR30-based gene knockdown.

Nucleic Acids Res. 34:e53 (2006); Development of miR155-based shRNA vectors.

J Gene Med. 9:620 (2007); Development of IPTG-inducible gene knockdown system.

Proc Natl Acad Sci USA. 101:10380 (2004); Development of Cre-lox-regulated gene knockdown system.

Q&A

What are the pros and cons of shRNA-mediated knockdown versus CRISPR- or TALEN-mediated knockout?

Either shRNA-mediated knockdown or nuclease-mediated knockout (e.g. CRISPR or TALEN) can be valuable experimental approach to study the loss-of-function effects of a gene of interest in cell culture. In order to decide which method is optimal for your specific application, there are a few things you should consider.

Mechanisms

  • Knockdown vectors: knockdown vectors express short hairpin RNAs (shRNAs) that repress the function of target mRNAs within the cell by inducing their cleavage and repressing their translation. Therefore, shRNA knockdown vectors are not associated with any DNA level sequence change of the gene of interest.
  • Knockout vectors: CRISPR and TALEN both function by directing nucleases to cut specific target sites in the genome. These cuts are then inefficiently repaired by the cellular machinery, resulting in permanent mutations, such as small insertions or deletions, at the sites of repair. A subset of these mutations will result in loss of function of the gene of interest due to frame-shifts, premature stop codons, etc. If two closely positioned cut sites in the genome (i.e. within several kb) are targeted simultaneously, this can also result in the deletion of the intervening region.

Effectiveness

shRNA-mediated knockdown will never completely repress the expression of the target gene. Even for the most effective shRNAs, some residual expression of the target gene will remain. In contrast, in a fraction of treated cells, CRISPR and TALEN can generate permanent mutations which may result in complete loss of gene function.

Consistency and uniformity

shRNA vectors generally provide high cell-to-cell uniformity within the pool of treated cells and very consistent results between experiments. In contrast, CRISPR and TALEN produce results that are highly non-uniform from cell to cell due to the stochastic nature of the mutations introduced. To fully knock out the gene of interest in a cell, all copies of the gene in the cell must be knocked out. Given that normal cells have two copies of any gene (except for X- or Y-linked genes) while cancer cells can have more than two copies, such full knockout cells may represent a very small fraction of all the treated cells. For this reason, nuclease-mediated knockout experiments require the screening of clones by sequencing to identify the subset in which all copies of the gene of interest have been knocked out.

Off-target effects

Off-target effects have been reported for both shRNA-mediated knockdown and nuclease-mediated knockout. The off-target phenotype(s) can be estimated by using multiple different shRNAs to target the same gene. If a gene knocked down by multiple different shRNAs results in consistent phenotype(s), then it argues against the phenotype(s) being caused by off-target effects. For CRISPR- or TALEN-mediated knockout, multiple clones containing loss-of-function mutations should be analyzed in order to account for any phenotype(s) that may be due to off-target mutations. Additionally, bioinformatically identified off-target sites could be sequenced in the clones to see if they have been mutated.

相同組み換え用のドナーベクターをデザインする
なぜshRNAを使った目的遺伝子のノックダウンがワークしないことがあるのですか? 
 

全てのshRNAが効果的にワークするわけではありません

VectorBuilderの経験と、ユーザーからのフィードバックを総合すると、一般的にある遺伝子に対して 3 ー4 種類のshRNAを試した場合、 2-3種類のshRNAで満足いくノックダウン効果が得られていることがわかっています。しかし選択した全てのshRNAがワークするわけではないことは必ず念頭に置いてください。一般的に、~50-70% のshRNAがノックダウンの表現型を表し、そのうちの ~20-30% は強いノックダウン効果を表します。仮に1-2種類のターゲットshRNAシークエンスを特定の1遺伝子に試した場合、チャンスとしては満足いくノックダウン効果が観察されないこともあり得ます。その場合、検証された別のshRNAを試すことが勧められます。複数の異なるshRNAをミックスした“カクテル” shRNAを試す場合もあり、ノックダウン効率の改善に役立つこともあります。

目的遺伝子のノックダウン効果を検証するアッセイ系が正しく実施できていない

最も一般的で感度の高いshRNAノックダウンの評価アッセイ系はRT-PCRです。複数のプライマーペアーを試し、最も特異性の高いプライマーペアーを使うことをお勧めします。一般的に、RT-qPCRのプライマーは、exon-exonのジャンクションシークエンスを挟むことで、サンプルにコンタミしたゲノム由来のDNAからの増幅を避けるようにデザインします。新しいプライマーペアーを試す際は、アガロースゲル電気泳動でバンドサイズを確認したり、サンガーシークエンスで増幅したPCR産物の確認を行うこともあります。RT-PCRには、必ずminus-RTコントロールを取って、ゲノムDNAのコンタミ率を概算できるようにしてください NCBI primer designing tool はデザインしたプライマーをコンピューター上(in silico)で検証できる良いツールです。

ウエスタンブロッティングもノックダウン効率検証のよいアッセイ系です。弱点は使用する抗体によっては非特異的な偽陽性バンドが出てしまい、結果を見誤るリスクがあることです。ウエスタンブロッティングに使う抗体は、目的遺伝子産物を特異的に認識することを予め確認してから使用してください。

shRNAが目的遺伝子の転写アイソフォームの一部にしかターゲッティングできていない

shRNAをデザインする際、目的遺伝子にisoformが存在する場合は、できる限り多くのisoformをターゲットできるようにデザインすることをお勧めします。 反対に、特定のisoformのノックダウンでは、可能な限り特異性の高いターゲットを選択してください。VectorBuilderは、特定の動物種に対して、最適化したshRNAデータベースを確立しています。shRNAベクターをご自分でデザインし、VectorBuilderに受託構築をご依頼いただく際は、VectorBuilderのデータベースをお試しになられることをお勧めします。このデータベースには、全てのshRNAのリスト、シークエンス、スコアーが一覧表示され、かつUCSC Genome Browserへのリンクからゲノムシークエンスのどの位置にターゲットシークエンスが位置するか確認することができます。

shRNAノックダウンスコアーはどのように決定されていますか? 

VectorBuilderのshRNAは the RNAi consortium (TRC) のアルゴリズムをベースとして改良したshRNAデザインとスコアーを採用しています。各RefSeq に登録されている転写産物に対して、ターゲットサイトの候補となる21merシークエンスが全て同定されています。候補ターゲットサイトは、ノックダウン効率や特異性を低下させるような特徴が検出されたり、クローニング不可性 (同一塩基≥4 連続、G または C塩基の≥7連続、GC 含量<25% またはGC含量>60%、そして 5’末端にAA )が検出された場合、候補から除外されます。ノックダウンスコアーは、内部にステムループ構造を持つ、3’末端に向てGC含量が高くなる、既知の miRNA シードシークエンス、他の遺伝子に対するオフターゲットマッチなどが検出されるとペナルティーをつけ、スコア化しています。また、1遺伝子に対して複数の転写産物が同定されている場合(alternative transcripts)、全ての転写産物に対して、最も高いスコアーを出すターゲットサイトを選び出しています。

全てのスコアーは ≥0以上の数字で、中間値(mean)が ~5、そして標準偏差が ~5となっています。95% のshRNAでスコアーは≤15となっています。例えば、あるshRNAのノックダウンスコアーが15であるとすると、最も効果の高いノックダウンパフォーマンスが予想され、クローニングも可能と判断されます。一方、ノックダウンスコアーが0のshRNAでは、最悪のノックダウンパフォーマンス、またはクローニング不可能なターゲットシークエンスと判断されます。 

ただ、ノックダウンスコアーはあくまでも予測シークエンスです。実際のノックダウン効率はスコアーからかけ離れている場合もあるかもしれません。低いノックダウンスコアーが与えられたターゲットシークエンスでも、shRNAとして研究論文につかわれていて、比較的よくワークしている場合もあります。また、3'UTR領域のターゲットでも、コーディング領域のターゲットの場合と同様に効果があることも念頭に置いてください。