Establishment of recombinant adeno-associated virus carrier production process
A virus vector and production process technology, applied in the field of establishment of recombinant adeno-associated virus vector production process, can solve the problems of many influencing factors, complicated operation, low immunogenicity, etc., and achieve stable and lasting expression, low immunogenicity, high purity effect
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Embodiment 1
[0048] Example 1 Preparation of rAAV vector particles
[0049] Such as image 3 Shown, the preparation method of a kind of rAAV vector particle in this implementation comprises the following steps:
[0050] (1) Culture of HEK293T cells:
[0051] Production of rAAV vector particles was performed in HEK293T. HEK293T cells were mixed at 1.4×10 per dish 6 The amount of cells was inoculated in a 10cm culture dish containing 15% (volume percent concentration) FBS DMEM medium, and placed at 37 ° C, 5% CO 2 cultured in an environment of saturated humidity. Transfection was performed 2 days after inoculation.
[0052] (2) Transfection of HEK293T cells:
[0053] Before transfection, the cell culture medium was replaced with 5 ml of DMEM medium containing 5% (volume percent concentration) FBS. The two helper vector plasmids pHelper( Figure 1A ), prAAV-RC ( Figure 1A ) and a shuttle vector plasmid prAAV-GFP ( Figure 1B ) were co-transfected into HEK293T cells at a mass ratio of...
Embodiment 2
[0060] Example 2 Preparation of rAAV vector particles
[0061] Such as image 3 Shown, the preparation method of a kind of rAAV vector particle in this implementation comprises the following steps:
[0062] (5) Culture of HEK293T cells:
[0063] Production of rAAV vector particles was performed in HEK293T. HEK293T cells were mixed at 1.4×10 per dish 6 The amount of cells was inoculated in a 10 cm culture dish containing 8% (volume percent concentration) FBS DMEM medium, and placed at 37 ° C, 5% CO 2 cultured in an environment of saturated humidity. Transfection was performed 2 days after inoculation.
[0064] (6) Transfection of HEK293T cells:
[0065] Before transfection, the cell culture medium was replaced with 5 ml of DMEM medium containing 2% (volume percent concentration) FBS. The two helper vector plasmids pHelper( Figure 1A ), prAAV-RC ( Figure 1A ) and a shuttle vector plasmid prAAV-GFP ( Figure 1B ) were co-transfected into HEK293T cells at a mass ratio of...
Embodiment 3
[0072] Example 3 Preparation of rAAV vector particles
[0073] Such as image 3 Shown, the preparation method of a kind of rAAV vector particle in this implementation comprises the following steps:
[0074] (9) Culture of HEK293T cells:
[0075] Production of rAAV vector particles was performed in HEK293T. HEK293T cells were mixed at 1.4×10 per dish 6 The amount of cells was inoculated in a 10cm culture dish containing 20% (volume percent concentration) FBS DMEM medium, and placed at 37 ° C, 5% CO 2 cultured in an environment of saturated humidity. Transfection was performed 2 days after inoculation.
[0076] (10) Transfection of HEK293T cells:
[0077] Before transfection, the cell culture medium was replaced with 5 ml of DMEM medium containing 10% (volume percent concentration) FBS. The two helper vector plasmids pHelper( Figure 1A ), prAAV-RC ( Figure 1A ) and a shuttle vector plasmid prAAV-GFP ( Figure 1B ) were co-transfected into HEK293T cells at a mass rati...
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