BPS Bioscience的AAV定制化服务

Introduction

Adeno-Associated Viruses (AAV) are useful for gene therapy due to their gene transfer efficiency and safety. BPS Bioscience is developing a portfolio of AAV products to support your research needs. In addition, our custom services team will accelerate the design and production of your favorite AAV vectors.

Recombinant AAVs can be used in a Biosafety Level 1 (BSL-1) facility. Inside the cell, AAVs are episomal and can result in the sustained expression of a gene of interest for up to 6 months in non-dividing cells and up to 2 months in dividing cells HEK293.

AAV has at least 11 naturally-occurring serotypes. Each serotype shows preferential binding for specific cell types and tissues. Scientists can use this tropism to efficiently target specific cell types.

Our AAV team will design your custom AAV constructs and will manufacture ready-for-use viral particles to transduce your gene(s) of interest. Applications include CRISPR-mediated genetic engineering, protein expression, and the design and optimization of AAV-mediated gene transfer using reporter AAVs.

AAV Reporter Vectors

Reporter proteins, such as luciferase or fluorescent markers, are ideal to visualize and/or quantify gene expression following AAV transduction. Luciferase, ZsGreen, and mCherry-containing AAVs can be used to optimize transduction and experimental conditions, track transgene expression over time, or be used as internal controls. BPS Bioscience offers a selection of AAV Reporter vectors. If your ideal vector is not in the list, we can build it for you.

AAV Reporter Vectors Produce Robust Signals After Transduction

A: 4-20% SDS-PAGE gel staining showing purified AAV1-ZsGreen particles. The protein ladder is in lane 1 and AAV1 is in lane 2. The gel was stained using One-Step Lumitein™ UV Protein Gel Stain (Biotium) and imaged using the used an ethidium bromide filter. The image was reversed to show protein bands in black.

B: Transduction of HEK293 cells using AAV1-ZsGreen. Expression of ZsGreen in the target cells was observed under a fluorescence microscope 72 hours after transduction. ZsGreen expression was stable over time and still observed 30 days after transduction.

A: Transduction of HEK293 cells with an AAV-Luciferase. Luciferase activity was measured using the ONE-Step™ Luciferase Assay System (BPS Bioscience #60690) 72 hours after transduction.

B: Transduction of HEK293 cells with an AAV-mCherry. The expression of mCherry was observed under a fluorescence microscope 72 hours after transduction.

CRISPR/Cas9 AAV Vectors

Cas9 is an endonuclease enzyme that is recruited to a specific DNA sequence by the sgRNA (single guide RNA) to introduce a double stranded break into the DNA. In mammalian cells this break is repaired either through Non-Homologous End Joining (NHEJ) or Homologous Recombination. NHEJ often results in the deletion or insertion of several base pairs at the cut site, which, when resulting in a frameshift, causes the functional inactivation of the gene.

SaCas9 (Staphylococcus aureus CRISPR associated protein 9) has demonstrated high cutting efficiency in mammalian cells, and its smaller size makes it ideal for packaging into AAV. These AAV-SaCas9 vectors are used to generate a SaCas9 over-expressing cell line for knockout or knock-in studies.

We also offer custom AAV production services to package the AAV-SaCas9 with your gene-specific sgRNA in the same virions.

Transduction of HEK293 cells using an AAV-SaCas9.
Cells were transduced with AAV-Cas9 virions at an
MOI of 1 x 10⁴. 72 hours after transduction, SaCas9
expression in the target cells were detected by
Western blot with an anti-HA.11 antibody. GAPDH
was used as a loading control.

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