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miRNA Expression and Inhibition

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miRNA Expression Vectors

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Human microRNA (hsa-mir) precursors and approximately 100-bp upstream and downstream flanking genomic sequences were PCR amplified and cloned into a self-inactivated (SIN) lentiviral vector to generate pLV-miRNA vectors (Fig. 1). The cloning site of pre-miRNA genomic fragments is within the intron of human EF1a promoter region. Our pLV miRNA vector is driven by the RSV-LTR chimeric promoter, and can be used in conjunction with either 2nd or 3rd generation packaging mixes to generate your lentivirus. The miRNA of interest can be delivered into cells by transient transfection of the pLV-miRNA plasmid or lentiviral transduction, while the miRNA lentiviral stock is prepared from cotransfecting HEK 293T cells with the pLV-miRNA plasmid and lentiviral packaging vector mix. Our miRNA expression systems provide several advantages to researchers:

Figure 1. Lentiviral vector for miRNA expression.
  • Our pLV-miRNA plasmid is an optimized vector system for miRNA expression.

    Unlike other available miRNA expression vectors, the miRNA precursor is cloned into the intron of human housekeeping gene EF1a promoter region. Therefore, the process of miRNA in intron minimizes impact on expression of selection marker. Since the miRNA precursor and the selection marker are in the same transcription unit, miRNA transcriptional activity can be easily monitored.

  • Lentiviral transduction is one of the most effective delivery systems to express miRNA, shRNA, cDNA, etc.

    Unlike the retroviral system, the lentiviral integration is cell cycle independent. The genetic materials encoded by the lentivirus can be efficiently delivered into both dividing and non-dividing cells.

  • Our lenti-miRNA viral genome is integrated into its host chromosome.

    Biosettia’s lenti-miRNA is integrated into its host chromosome, thus allowing it to be stably expressed in transduced cell lines.

  • Our human EF1a promoter is unlikely to be silenced in cells.

    It has been reported that the activities of some viral promoters such as CMV and SV40 are potentially silenced by DNA methylation after a period of time. The human EF1a promoter, used to express miRNA precursors and the puromycin selection marker, is a house-keeping gene promoter. Therefore, it is unlikely to be silenced by methylation in vitro and in vivo.

  • Our rPuro gene product, expressed from the EF1a promoter, is the red fluorescent puromycin-N-acetyl-transferase.

    Model cells transduced by lenti-miRNA can not only be selected in the presence of puromycin in the medium, but also display red fluorescence at excitation/emission wavelengths of 587/610 nm (Fig. 2).

    Figure 2. Microscopy images of 293T cells transduced with hsa-mir-24-1 lentivirus.
  • Each miRNA precursor in the lentiviral vector was cloned from its native context, including its flanking and stem-loop precursor sequence.

    This ensures that each miRNA is properly expressed and processed, and would function similarly to its endogenous form.

miRNA Expression Lentiviruses

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Biosettia has cloned over 600 of the most popular human microRNA precursors into self-inactivated (SIN) lentiviral vectors to generate a comprehensive pLV-miRNA collection. In addition to our lentiviral miRNA vector products, we provide ready-to-use lentiviral stock prepared from our own plasmids and lentiviral packaging mix in 1 ml tubes online. For researchers interested in our high-titer and high-volume lentivirus, please contact info@biosettia.com to request a quote.

Figure 3. Lentivirus expresses miRNA cloned in the EF1a intron.

Product Components

  • 1ml Lentivirus (1 x107 infection units per ml (IU/ml) in HT1080 cells)
  • 10ml Lentivirus (Not sold online, please inquire about availability of this product)

General Preparation of our Lentiviral Stock:

  • Cotransfection of HEK 293T cells with the lenti-miRNA vector and plasmids expressing Gag-Pol gene products and the vesicular stomatitis virus envelope G (VSV-G).
  • Collection of lentiviral supernatants at 48 hours post transfection and stored at -70ºC.
  • The titer of our virus is generally above 1 × 107 infection units per ml (IU/ml) in HT1080 cells. Please note that virus titer varies among cell lines.

Shipping & Handling of our Lentivirus:

  • All of our miRNA available online are ready-to-use and in stock. Please note that producing high titer or high volume lentivirus may require longer turnaround time.
  • Biosettia ships all lentivirus products in dry ice via overnight delivery. For all orders delivered outside the US, extra dry ice is needed to ensure your virus is safe from any customs delay. Please note that additional shipping fees may apply to your order.
  • Please store at -70 Degrees Celsius upon arrival, and allow our lentiviral products to thaw slowly on ice just before use. For more information about handling lentivirus, please visit our protocol page.

Vectors for Inhibition of miRNA

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Studies have shown that the function of miRNA in repressing endogenous gene expression can be effectively inhibited by the artificial miRNA binding sites expressed from plasmids or viruses in cells. miRNA inhibitors can be divided into two categories:

— Chemically synthesized, single-stranted oligonucleotides, such as 2′-O-methylated antisense RNA and locked nucleic acids (LNA)

— Multiple copies of a miRNA binding site encoded in an RNA transcript expressed from DNA vectors or recombinant viruses.

Biosettia’s miRNA inhibitor (miR-Locker) combines both strategies to contain two copies of single stranded nucleotides, each being perfectly complementary to the 5′ and 3′ ends of your target miRNA with a bulge between each region. These miR-Lockers are expressed from a Biosettia lentiviral expression system (pLV-miR-Locker), which effectively and stably delivers the miR-Locker sequences into cells, and allows continuous overexpression of the competitive inhibitors for long-term miRNA inhibition.

Figure 3. The miR-Locker sequence is present on both transcripts expressed from the EF1a and the mouse U6 promoters, designated as transcript 1 and transcript 2, respectively.

An expression cassette containing the miR-Locker sequence (two copies of a miRNA binding site) driven by a mouse U6 promoter (mU6) is cloned into the 3 prime UTR of a GFP-Bsd reporter gene driven by an EF1a promoter, downstream of the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), to generate our pLV-miR-Locker vector.

Figure 4. Plasmid map of our pLV-miR-Locker vector

Shipping & Handling of our Lentiviral Vectors:

  • — All of our miRNA lockers are made to order and may require up to one week to prepare.
  • — We ship our plasmids via overnight delivery in glycerol stock at room temperature. This allows our plasmids to remain stable at room temperature until receipt. Please note that plasmids should be stored at -70 Degrees Celsius upon arrival. Please be aware that an additional shipping fee may be applied to all orders.
  • — For more information about handling our miRNA inhibition vectors, please visit our protocol page.

miRNA Custom Services

Biosettia owns one of the largest collections of miRNA available. This library includes over 600 human miRNA precursors cloned into intronic regions of E1a-RFP plasmids and are available for near immediate shipment to help expedite the process of your experiments. In addition to making these plasmids available to you, Biosettia’s facilities are capable of providing a host of custom services relating to miRNA expression and inhibition, including:

Creating custom miRNA expression vector constructs

Generating miRNA mutants

Reversing miRNA precursors within Biosettia’s expression vector

Carrying out miRNA reporter assays

Validating potential miRNA-target candidates

Generating large volume / high titer miRNA lentivirus

Inhibiting mature miRNA via miR-locker constructs

miRNA functional screening

To inquire about these services or to request a quote, please e-mail us at info@biosettia.com

miRNA Plasmid Maps & Sequences

miRNA Expression Vectors miR Locker Vectors