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Custom-made shRNA Lentivirus Particle (SH4001)

Availability: In stock

USD$460.00

Quick Overview

Custom-made shRNA Lentivirus Particle (SH4001) products from ATCGbio Life Technology are recombinant shRNA expression lentivirus particle to be ready to infect directly for knocking-down gene of interest. It’s specifically designed for scientists who don’t have time or don’t prefer to make lentivirus by themselves. It’s the easiest way to knockdown gene of interest.
Customer can choose the region of knocking-down (CDS or 3'-UTR), reporter co-expression (GFP, RFP or iRFP) and antobiotics resistance (puromycin, hygromycin, blasticidin or zeocin).
The Product Components:
1. Custom-made shRNA Lentivirus particle 1(SH4001-XXX1), 9.0ml
2. Custom-made shRNA Lentivirus particle 2 (SH4001-XXX2), 9.0ml
3. shRNA Lentivirus particle (control, SH3002), 9.0ml
4. Storage solution(reconstitution solution, SH3003), 1.0ml
The total price for the first order is $460.00. From the second time order, the same target (one of two lenti particles) and control only cost $218.00.
Please CONTACT US by fill-in your fist-time order number, date and contact information including name, email, phone and address. We will provide you a second order code.
Order
Please submit the information into the following box for order:
1. An official symbol and gene ID or Accession number (including isoforms number)
2. Region of knocking-down (CDS or 3'-UTR)
3. Reporter co-expression: GFP, RFP or iRFP
4. Antibiotics resistance: puromycin, hygromycin, blasticidin or zeocin
If you don't sepcify for 2, 3, and 4, we'll offer you a viral particle expressing shRNA for target gene at CDS or 3'-UTR, GFP co-expression and hygromycin resistance.
If your target genes are 2 or more, please enter the number of your target genes (not the number of the isoforms) in the "Qty" box before you click "ADD TO CART".
We may contact after feasibility evaluation.

Custom-made shRNA Lentivirus Particle (SH4001)

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USD$460.00

Details

Download: User Manual PDF | MSDS PDF

The product contains 4 components:

 Catalog   Components Size    Shipping and Storage
 SH4001-XXX1   Custom-made shRNA Lentivirus particle 1
9.0ml ×1   Shipped at room temperature, store at -20 °C
 SH4001-XXX2   Custom-made shRNA Lentivirus particle 2
9.0ml ×1   Shipped at room temperature, store at -20 °C
 SH3002   shRNA Lentivirus particle (control) 9.0ml×1   Shipped at room temperature, store at -20 °C
 SH3003   Lenti-Storage Solution(reconstitution) 1.0ml ×1   Shipped at room temperature, Keep at 4°C

The highlight features of the products are:
1. Most advanced shRNA design.No need to add polybrene.Near 100% transduction (Fig.1.).
2. High infectious efficiency(Fig.2.), up to 0.9 x 108 TU/ml.
3. Co-express GFP protein and hygromycine resistance gene.
4. Just add and incubate overnight (16 hrs).
5. No cytotoxicity. Injectable to small animal without immonoreactive symptoms.
6. Unlike siRNA, transduced cells show knock-down effect in both dividing and nondividing cells.
7. Cell-line can be made in a week.
8. Suitable for both primary cells and transformed cells.
Lentivirus particle will be shipped in propriety solution making high transduction without polybrene and unprecedented stability:
• Virus solution can be stored at -20 °C.
• Allow freezing and thawing up to 2 times.
• Be stable in room temperature at least 3-4 days.
• If necessary (the cells are incompatible with DMEM base medium), purification is easy as just centrifuge for 30min at 1500 × g.
Custom-made shRNA Lentivirus particle products from ATCGbio Life Technology are created base on safer 3rd. generation recombinant lentivirus vector through our new shRNA design system; and produced by our own pLenti™ Lentivirus Production Kit (LT1001). Virus expresses shRNA for target gene, GFP protein and hygromycine resistance gene product. It is suitable for both cultured cells and in vivo injection to small animals.  Lentivirus solution is DMEM based medium containing 2% FBS and our lentivirus precipitation/purification solution. It does not contain any chemicals used to boost lentivirus titers such as chloroquin, acetate or nicotines. The solution can be used directly to the cells growing in DMEM base medium without further precipitation/purification. There is no visible viral toxicities (e.g. change in cell morphology or/and cell detachment from culture plate) if the viral solution added up to a half amount of cell culture medium as described in table 1. Some special cells such as keratinocytes may be used to grow with special medium which is incompatible with DMEM or FBS. In such a case, it is recommended to precipitate/purify lentivirus and reconstitute it with storage solution as described in User Manual.

              Table.1.

                           Size of Culture          Culture Medium         Lentiviral Solution      
  1 well of 6-well plate 1.0ml 0.5ml
  1 well of 12-well plate 0.5ml 0.25ml
  1 well of 24-well plate 0.25ml 0.125ml

 

Fig.1. HepG2 cells grown in 100µl MEM+10% FBS on the 96-well plate were infected with 50µl shRNA lentivirus solution 48hrs.
Near 100% transduction.
     
Fig.2. 64hrs after infection without antibiotic selection, the target gene mRNAs were down to 28.8% in average of 10 shRNA targets in HepG2 cells.
More details are described below.
HepG2-celld-were-infected-with-50µl-shRNA-lentivirus-solution-48hrs            
     

shRNA-lentivirus-performance                                           Non-target Gene-specific                           shRNA        shRNA

As shown in Figure 2. Ten transcription factors expressed in HepG2 cells were selected for this experiment. The lentivirus plasmids expressing shRNA were made for each target gene using our propriety design scheme (One shRNA sequence was selected for one target in each design). Lentivirus was created in 12-well plates using our pLenti™ Lentivirus Production Kit (LT1001), which produces a high titer stable lentivirus solution in 48hrs. HepG2 cells were grown in 96-well palate and infected with each virus including negative control shRNA by adding 50µl crude virus solution into HepG2 cells cultured with100µl culture medium for overnight(16hrs.). The cells were incubated further 48hrs., and harvested for Direct RT real-time PCR.  As shown in the right figure, 64 hrs. after infection, target gene mRNAs were down to 28.8% in average of 10 shRNA targets (p<0.0001 by paired t-test). 9 out of 10 design shRNA were able to suppress more than 70%, and only one out of 10 shRNA suppressed more modestly about 60%. Suppression will be greater, when the cells would be cultured longer time with antibiotics selection.
The experiment results of lentivirus production and infection (lentivirus produced by pLenti™ Lentivirus Production Kit (LT1001)):

Fig.A and Fig.B (magnification 100x): GFP expressing lentivirus was created with two different medium

    Conventional DMEM+10% FBS                              Our Lentivirus Production Medium + 2% FBS

 Lentivirus-infect293T-cell(ordernary-medium-cell-culture) Fig.A  

       Lentivirus-infect293T-cell(our-Production-Medium-culture) Fig.B       

50µl of 3ml crude lentivirus with medium was directly used to infect 293T cells (without polybrene) grown in a separate 6 well plate. 48 hours later the expression of GFP was assessed.

Fig. C and Fig. D (magnification 100x): shRNA experssion lentivurs was produced by the pLenti™ Lentivirus Production Kit (LT1001); and infected to HUVEC (Human Umbilical Vein Endothelial Cells).

Primary human umbilical vein endothelial cells (HUVEC) growing in 100mm dishes were infected by lentivirus shRNA negative control co-expressing with GFP. GFP expression were observed 3 days after infection.

Lentivirus-infection-in-human-endothelial-cells1-3br.jpg Fig.C

    Lentivirus-infection-in-human-endothelial-cells1-3fl.jpg Fig.D
   Fig.E (magnification 100x)            Lentivirus-in-vivo-example(fat-injection)                          

Fig.E: In vivo transduction sample

50 µl of purified lentivirus with our kit was injected into a mouse subcutaneous adipose tissue (without polybrene). After three days, the tissue was harvested and observed under fluorescence microscope and bright field.

                                        

References

  1. Y. Ido, et al. PLoS ONE, 2012 Apr 07(4): e35092
  1. Lan F, et al.  J Biol Chem. 2008 Oct 10;283(41):27628-35

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