1. Adeno-associated virus (AAV) is a small DNA virus that requires assistance from other viruses like adenovirus to replicate. It is commonly used as a vector for gene delivery due to its low immunogenicity.
2. Recombinant AAV (rAAV) vectors are produced using a 3-plasmid system in AAV-293 packaging cells. This results in viral particles containing the gene of interest.
3. Purification methods like iodixanol gradient ultracentrifugation are used to separate rAAV from contaminants before concentration through ultrafiltration.
AAV production protocol:packaging,concentration and purification-gene medissuser8cc395
1. Adeno-associated virus (AAV) is a small DNA virus that does not cause disease on its own but requires assistance from other viruses like adenovirus to replicate. Recombinant AAV (rAAV) vectors are commonly used for gene delivery by replacing the viral genome with a transgene cassette.
2. Production of rAAV involves co-transfecting three plasmids (containing the transgene, Rep/Cap genes, and adenoviral genes) into AAV-293 packaging cells. Harvested viral particles are purified using iodixanol gradient ultracentrifugation.
3. Purified rAAV is concentrated and quality is assessed by detecting the
This document provides protocols for producing recombinant adeno-associated virus (rAAV) vectors. It first describes AAV biology, noting that AAV requires helper viruses to replicate and has nonpathogenic wildtype strains. The protocol then outlines a three-plasmid system to generate rAAV using AAV-293 packaging cells. This involves transfecting cells with vectors encoding the gene of interest flanked by AAV inverted terminal repeats along with helper plasmids. Harvested rAAV is purified using iodixanol gradient ultracentrifugation and concentrated using ultrafiltration to remove contaminants. Quality control assesses the presence of three AAV capsid proteins to confirm vector purity.
GeneMedi-Adenovirus production protocol-packaging concentration and purificationMelissaZHANG18
1. The document describes a protocol for producing recombinant adenovirus using an adenovirus expression system. Key steps include cloning the gene of interest into an adenovirus shuttle vector, co-transfecting the shuttle vector and adenovirus backbone into 293A packaging cells, identifying viral plaques, and amplifying and purifying the recombinant adenovirus.
2. Recombinant adenovirus is produced via homologous recombination between the adenovirus shuttle vector containing the gene of interest and adenovirus backbone vector in 293A cells. Small viral plaques are picked and amplified through multiple passages.
3. The purified recombinant adenovirus is obtained through PEG precipitation, CsCl gradient
Recombinant adenovirus (rAdV) is a gene transfer vector that can deliver gene sequences into cells without genome integration. rAdV has advantages such as a large transgene capacity, ability to infect dividing and non-dividing cells, and high efficiency. rAdV is commonly produced using packaging systems like AdEasy and AdMAX, which introduce the gene of interest into a shuttle vector that is recombined into a viral backbone in 293A packaging cells. Plaques are selected and amplified through multiple passages to generate high-titer virus stocks for experiments.
Unveiling the Potential of your AAV Gene Therapy: Orthogonal methods to under...Merck Life Sciences
Watch the presentation of this webinar here: https://bit.ly/3pCCjPF
Ensure your Adeno-Associated Virus (AAV) is safe throughout its entire drug development journey. Learn methods that will help you speed to clinic, potentially treating diseases sooner and with greater effectiveness.
The potential of gene therapies to cure previously untreatable diseases has spurred the development of novel drugs, including those based on Adeno-Associated Virus (AAV). As with all biopharmaceuticals, it is important to identify and monitor the critical quality attributes (CQAs) of these products to ensure their safety and efficacy.
In this webinar, we will present a range of orthogonal methods to understand and define the CQAs of AAV products. These include assays for the confirmation of capsid protein identity and quantity, as well as the characterization of important product-related impurities, such as aggregates. Together these methods represent a comprehensive analytical testing package to support the characterization and lot release of AAV products.
In this webinar, you will learn:
• How to identify and monitor the critical quality attributes (CQAs) of your AAV therapy
• What assays to utilize to confirm capsid protein identity and quantity
• Why you need look to product characterization to identify and remove important product-related impurities
Unveiling the Potential of your AAV Gene Therapy: Orthogonal methods to under...MilliporeSigma
Watch the presentation of this webinar here: https://bit.ly/3pCCjPF
Ensure your Adeno-Associated Virus (AAV) is safe throughout its entire drug development journey. Learn methods that will help you speed to clinic, potentially treating diseases sooner and with greater effectiveness.
The potential of gene therapies to cure previously untreatable diseases has spurred the development of novel drugs, including those based on Adeno-Associated Virus (AAV). As with all biopharmaceuticals, it is important to identify and monitor the critical quality attributes (CQAs) of these products to ensure their safety and efficacy.
In this webinar, we will present a range of orthogonal methods to understand and define the CQAs of AAV products. These include assays for the confirmation of capsid protein identity and quantity, as well as the characterization of important product-related impurities, such as aggregates. Together these methods represent a comprehensive analytical testing package to support the characterization and lot release of AAV products.
In this webinar, you will learn:
• How to identify and monitor the critical quality attributes (CQAs) of your AAV therapy
• What assays to utilize to confirm capsid protein identity and quantity
• Why you need look to product characterization to identify and remove important product-related impurities
The document provides information about adenovirus (AdV) including storage, dilution, introduction, products, services, vectors, and experimental procedures for AdV production. The key steps are:
1) Transfecting virus plasmids into 293A packaging cells to produce initial virus particles.
2) Observing plaque formation and collecting isolated plaques to amplify virus through multiple passages.
3) Purifying and concentrating the amplified virus for use in transduction experiments and animal studies.
AAV production protocol:packaging,concentration and purification-gene medissuser8cc395
1. Adeno-associated virus (AAV) is a small DNA virus that does not cause disease on its own but requires assistance from other viruses like adenovirus to replicate. Recombinant AAV (rAAV) vectors are commonly used for gene delivery by replacing the viral genome with a transgene cassette.
2. Production of rAAV involves co-transfecting three plasmids (containing the transgene, Rep/Cap genes, and adenoviral genes) into AAV-293 packaging cells. Harvested viral particles are purified using iodixanol gradient ultracentrifugation.
3. Purified rAAV is concentrated and quality is assessed by detecting the
This document provides protocols for producing recombinant adeno-associated virus (rAAV) vectors. It first describes AAV biology, noting that AAV requires helper viruses to replicate and has nonpathogenic wildtype strains. The protocol then outlines a three-plasmid system to generate rAAV using AAV-293 packaging cells. This involves transfecting cells with vectors encoding the gene of interest flanked by AAV inverted terminal repeats along with helper plasmids. Harvested rAAV is purified using iodixanol gradient ultracentrifugation and concentrated using ultrafiltration to remove contaminants. Quality control assesses the presence of three AAV capsid proteins to confirm vector purity.
GeneMedi-Adenovirus production protocol-packaging concentration and purificationMelissaZHANG18
1. The document describes a protocol for producing recombinant adenovirus using an adenovirus expression system. Key steps include cloning the gene of interest into an adenovirus shuttle vector, co-transfecting the shuttle vector and adenovirus backbone into 293A packaging cells, identifying viral plaques, and amplifying and purifying the recombinant adenovirus.
2. Recombinant adenovirus is produced via homologous recombination between the adenovirus shuttle vector containing the gene of interest and adenovirus backbone vector in 293A cells. Small viral plaques are picked and amplified through multiple passages.
3. The purified recombinant adenovirus is obtained through PEG precipitation, CsCl gradient
Recombinant adenovirus (rAdV) is a gene transfer vector that can deliver gene sequences into cells without genome integration. rAdV has advantages such as a large transgene capacity, ability to infect dividing and non-dividing cells, and high efficiency. rAdV is commonly produced using packaging systems like AdEasy and AdMAX, which introduce the gene of interest into a shuttle vector that is recombined into a viral backbone in 293A packaging cells. Plaques are selected and amplified through multiple passages to generate high-titer virus stocks for experiments.
Unveiling the Potential of your AAV Gene Therapy: Orthogonal methods to under...Merck Life Sciences
Watch the presentation of this webinar here: https://bit.ly/3pCCjPF
Ensure your Adeno-Associated Virus (AAV) is safe throughout its entire drug development journey. Learn methods that will help you speed to clinic, potentially treating diseases sooner and with greater effectiveness.
The potential of gene therapies to cure previously untreatable diseases has spurred the development of novel drugs, including those based on Adeno-Associated Virus (AAV). As with all biopharmaceuticals, it is important to identify and monitor the critical quality attributes (CQAs) of these products to ensure their safety and efficacy.
In this webinar, we will present a range of orthogonal methods to understand and define the CQAs of AAV products. These include assays for the confirmation of capsid protein identity and quantity, as well as the characterization of important product-related impurities, such as aggregates. Together these methods represent a comprehensive analytical testing package to support the characterization and lot release of AAV products.
In this webinar, you will learn:
• How to identify and monitor the critical quality attributes (CQAs) of your AAV therapy
• What assays to utilize to confirm capsid protein identity and quantity
• Why you need look to product characterization to identify and remove important product-related impurities
Unveiling the Potential of your AAV Gene Therapy: Orthogonal methods to under...MilliporeSigma
Watch the presentation of this webinar here: https://bit.ly/3pCCjPF
Ensure your Adeno-Associated Virus (AAV) is safe throughout its entire drug development journey. Learn methods that will help you speed to clinic, potentially treating diseases sooner and with greater effectiveness.
The potential of gene therapies to cure previously untreatable diseases has spurred the development of novel drugs, including those based on Adeno-Associated Virus (AAV). As with all biopharmaceuticals, it is important to identify and monitor the critical quality attributes (CQAs) of these products to ensure their safety and efficacy.
In this webinar, we will present a range of orthogonal methods to understand and define the CQAs of AAV products. These include assays for the confirmation of capsid protein identity and quantity, as well as the characterization of important product-related impurities, such as aggregates. Together these methods represent a comprehensive analytical testing package to support the characterization and lot release of AAV products.
In this webinar, you will learn:
• How to identify and monitor the critical quality attributes (CQAs) of your AAV therapy
• What assays to utilize to confirm capsid protein identity and quantity
• Why you need look to product characterization to identify and remove important product-related impurities
The document provides information about adenovirus (AdV) including storage, dilution, introduction, products, services, vectors, and experimental procedures for AdV production. The key steps are:
1) Transfecting virus plasmids into 293A packaging cells to produce initial virus particles.
2) Observing plaque formation and collecting isolated plaques to amplify virus through multiple passages.
3) Purifying and concentrating the amplified virus for use in transduction experiments and animal studies.
1. Bovine papillomavirus (BPV) vectors utilize the circular, double-stranded DNA genome of BPV. The BPV genome contains early and late regions and can transform cells without integrating.
2. There are three main types of BPV vectors. All contain the transforming 69% fragment of BPV and bacterial sequences. One type inserts a gene of interest, another adds a stimulating gene, and the third uses the full BPV genome.
3. Transformation efficiency is highest with the full BPV genome due to an enhancer in the non-transforming region. Stimulating genes can replace this enhancer's function when parts of the genome are removed. BPV vectors provide amplified
The emerging CRISPR/Cas9 gene editing technology greatly accelerates the R&D process in life sciences. Here, we briefly introduce CRISPR/Cas9 and its delivery strategies.
See the Whole Picture: Using SV-AUC for Empty/Full AAV Capsid AnalysisMilliporeSigma
Watch this webinar here: https://bit.ly/31ZZM3n
Join this webinar for key insights on using the SV-AUC assay for empty/full analysis of your AAV viral vector. We’ll cover the technical requirements for this assay, data interpretation, and finally how this assay fits into the larger picture of AAV characterization.
Recombinant adeno-associated viruses (AAV) are widely used as gene transfer vectors. However, AAV production generates mixed populations of viral capsids containing either complete viral vector genome (full capsids); partially filled, and those lacking the viral genome (empty capsids). Sedimentation Velocity Analytical Ultracentrifugation (SV-AUC) offers a robust, accurate, and consistent method for characterizing empty/full AAV capsid composition. In this webinar we will review the key technical requirements for performing an AUC assay as well as analysis and data interpretation of the results generated.
In this webinar, you will learn:
• Regulatory expectations for empty/full analysis
• Key technical requirements for running an AUC assay and how to interpret the data from the results generated
• How the AUC assay fits into the larger picture of AAV characterization
See the Whole Picture: Using SV-AUC for Empty/Full AAV Capsid AnalysisMerck Life Sciences
Regulatory agencies require characterization of AAV particle composition, including measurement of empty and full capsids. Analytical ultracentrifugation (AUC) provides a method to determine the percentage of empty, partial, and full capsids in a sample and fits into a multi-method approach to characterize identity, purity, biological activity, and safety of gene therapy products. Monitoring product impurities such as empty particles decreases safety risks.
Genemedi aav(adeno associated virus) user manualssuser8cc395
This document provides information on adeno-associated virus (AAV) including safe use, storage, dilution, introduction, advantages for gene delivery and expression, AAV serotypes and tropism, Genemedi AAV products and services, overall experimental procedures for AAV production including vector construction, packaging, collection, purification, titer detection, cell and animal infection testing, and several case studies.
Adeno-associated virus (AAV) is a small, non-enveloped virus that was first discovered in 1965 as a contaminant in adenovirus isolates. It requires a helper virus like adenovirus or herpes virus to productively infect cells. AAV has a life cycle with both lytic and lysogenic phases - in the presence of a helper virus it undergoes lytic replication and production of new virions, while in the absence of helper virus it can integrate into the host genome and remain latent. Recombinant AAV (rAAV) vectors are used in gene therapy, containing a transgene in place of the viral genes, with only the inverted terminal repeats remaining to function in
Hepatitis C virus (HCV) is a positive-sense RNA virus that can cause hepatocellular carcinoma (HCV). HCV has an RNA genome around 9,600 nucleotides in length that is translated into a single polyprotein. HCV replicates in hepatocytes using its RNA genome and structural and non-structural proteins. Chronic HCV infection can lead to liver damage, inflammation, and over many years increases the risk of hepatocellular carcinoma through cellular changes and mutations caused by the virus and immune response.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
A comprehensive study of shuttle vector & binary vector and its rules of in ...PRABAL SINGH
Vector: A vector is a DNA molecule that has the ability to replicate autonomously in an appropriate host cell and into which the DNA fragment to be cloned is integrated for cloning
Recombinant viral vectors are genetic engineering tools commonly used for gene transfer purpose with high transfection efficiency and site specific gene insertion.
The document summarizes key information about Hepatitis C virus (HCV). It belongs to the Flaviviridae family of viruses which includes yellow fever, West Nile virus, and dengue virus. HCV is a small enveloped virus with a positive sense RNA genome approximately 9,600 nucleotides in length. The genome contains a single open reading frame flanked by untranslated regions, and codes for structural and non-structural proteins. HCV enters the liver cells and uses its RNA for both translation and transcription to replicate itself and cause infection through multiple steps.
The document discusses yeast molecular biology and genetics. It provides information on commonly used yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. It discusses yeast genome, life cycle, vectors, cloning, transformation methods, and applications including gene expression and making mutants.
PRODUCTION OF SEROTYPE 6-DERIVED RECOMBINANT ADENO-ASSOCIATED VIRUS IN SERUM-...Dr. Érica Schulze
This document describes research into producing recombinant adeno-associated virus (rAAV) using serum-free suspension cultures of HEK 293 cells. The researchers investigated the effects of different DNA, polyethyleneimine (PEI) transfection reagent, and cell concentrations on rAAV yields. They found that transfecting at a cell concentration of 3x10^6 cells/mL resulted in a 2.5-3.5 fold increase in infectious virus particles (IVP) and genomic particles (Vg) compared to 1x10^6 cells/mL. When testing different PEI:DNA ratios, IVP ranged from 1.19x10^8 to 1.03x10^9 IVP/
The document discusses various methods of transfection in animals. Transfection is the process of introducing nucleic acids into eukaryotic cells. It describes viral transfection using bacteria like Agrobacterium tumefaciens and viruses. Non-viral methods include chemical transfection using calcium phosphate, liposomes, polyamines. Mechanical transfection employs microinjection or particle bombardment. Common chemical methods are calcium phosphate precipitation, polyplexes, and liposomes/lipoplexes. Viruses used are retroviruses, adenoviruses, adeno-associated viruses. Bacterial and viral vectors allow for integration into the host genome while chemical and mechanical are often transient.
HCV was discovered in 1989 as a major cause of non-A, non-B hepatitis. It is a single stranded RNA virus of the Flaviviridae family. An estimated 130-170 million people worldwide are chronically infected, making it a significant global health burden.
HCV has an enveloped virion that is 50-80 nm in diameter with E1 and E2 glycoprotein spikes. Its positive-sense single stranded RNA genome is translated into a polyprotein that is cleaved into structural and non-structural proteins. The non-structural proteins NS3, NS4, NS5B are involved in replication and assembly of new virions.
Older treatments for HCV involved peg
This presentation summarizes research on characterizing RNA aptamers that recognize Regnase-1. It discusses how aptamers were selected through SELEX to bind Regnase-1 with high affinity. Structural modeling and docking simulations were performed to analyze the tertiary structures of Regnase-1 and identified aptamers, as well as their interactions. The results provide insight into how Regnase-1 regulates immune responses through mRNA decay mediated by aptamer binding.
This document discusses different types of cloning vectors. It describes that vectors are used to carry foreign DNA into host cells. There are two main types of transformation vectors - cloning vectors which are used to increase copies of cloned DNA fragments, and expression vectors which are used to express foreign genes as proteins. Some examples of commonly used cloning vectors include pBR322 and pUC18/19, while examples of expression vectors include pET28 and pRSET vectors. The document then discusses various properties desirable in vectors such as an origin of replication, antibiotic resistance genes, and regulatory elements. It describes different types of vectors including plasmid vectors, bacteriophage vectors, cosmids, BACs/YACs, and mini chromosomes.
Gene transfer in the liver using recombinant adeno-associated virusJonathan G. Godwin
This document describes methods for delivering gene transfer vectors to the liver using recombinant adeno-associated virus (rAAV). It discusses that intravenous injection of rAAV serotypes results in efficient transduction of the liver. The document provides protocols for preparing rAAV vector samples and delivering the vectors to mouse liver through lateral tail vein injection, retro-orbital sinus injection, or portal vein injection. It notes that tail vein injection is the most common method but retro-orbital sinus injection can be used as an alternative for young or neonatal mice. Precise vector dosing and injection technique are emphasized for achieving effective liver-directed gene transfer.
This document discusses different types of vectors that can be used for genetic engineering, including animal viruses, plant viruses, retroviruses, shuttle vectors, and binary vectors. It provides details on the structure and use of tobacco mosaic virus (TMV) as a plant viral vector, describing how foreign genes can be stably replicated and spread systemically in plants using this system. It also summarizes key features of yeast episomal plasmids (YEps), retroviral vectors based on murine leukemia virus, and the binary vector system used for plant transformation via Agrobacterium tumefaciens.
The document discusses the Octet system from Pall ForteBio for measuring biomolecular interactions. It provides advantages of the Octet system such as label-free detection, real-time kinetic data acquisition, high sensitivity and throughput. The system uses biotinylated ligands immobilized on biosensors to monitor interactions with analytes. The general test procedure is outlined as sample loading, baseline measurement, association and dissociation phases from which binding kinetics are determined.
Lateral flow immunoassay (LFIA) is a membrane-based technique that provides rapid, inexpensive, and easy-to-use detection of analytes in complex samples. Key advantages are low cost, ease of use without specialized equipment, and ability to provide results in minutes. LFIAs detect analytes using various formats, including sandwich assays that detect large analytes using two antibodies, and competitive assays that detect small analytes. LFIAs have applications in healthcare, agriculture, food/water quality testing, and more.
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1. Bovine papillomavirus (BPV) vectors utilize the circular, double-stranded DNA genome of BPV. The BPV genome contains early and late regions and can transform cells without integrating.
2. There are three main types of BPV vectors. All contain the transforming 69% fragment of BPV and bacterial sequences. One type inserts a gene of interest, another adds a stimulating gene, and the third uses the full BPV genome.
3. Transformation efficiency is highest with the full BPV genome due to an enhancer in the non-transforming region. Stimulating genes can replace this enhancer's function when parts of the genome are removed. BPV vectors provide amplified
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Watch this webinar here: https://bit.ly/31ZZM3n
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Recombinant adeno-associated viruses (AAV) are widely used as gene transfer vectors. However, AAV production generates mixed populations of viral capsids containing either complete viral vector genome (full capsids); partially filled, and those lacking the viral genome (empty capsids). Sedimentation Velocity Analytical Ultracentrifugation (SV-AUC) offers a robust, accurate, and consistent method for characterizing empty/full AAV capsid composition. In this webinar we will review the key technical requirements for performing an AUC assay as well as analysis and data interpretation of the results generated.
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• Key technical requirements for running an AUC assay and how to interpret the data from the results generated
• How the AUC assay fits into the larger picture of AAV characterization
See the Whole Picture: Using SV-AUC for Empty/Full AAV Capsid AnalysisMerck Life Sciences
Regulatory agencies require characterization of AAV particle composition, including measurement of empty and full capsids. Analytical ultracentrifugation (AUC) provides a method to determine the percentage of empty, partial, and full capsids in a sample and fits into a multi-method approach to characterize identity, purity, biological activity, and safety of gene therapy products. Monitoring product impurities such as empty particles decreases safety risks.
Genemedi aav(adeno associated virus) user manualssuser8cc395
This document provides information on adeno-associated virus (AAV) including safe use, storage, dilution, introduction, advantages for gene delivery and expression, AAV serotypes and tropism, Genemedi AAV products and services, overall experimental procedures for AAV production including vector construction, packaging, collection, purification, titer detection, cell and animal infection testing, and several case studies.
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Hepatitis C virus (HCV) is a positive-sense RNA virus that can cause hepatocellular carcinoma (HCV). HCV has an RNA genome around 9,600 nucleotides in length that is translated into a single polyprotein. HCV replicates in hepatocytes using its RNA genome and structural and non-structural proteins. Chronic HCV infection can lead to liver damage, inflammation, and over many years increases the risk of hepatocellular carcinoma through cellular changes and mutations caused by the virus and immune response.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
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Vector: A vector is a DNA molecule that has the ability to replicate autonomously in an appropriate host cell and into which the DNA fragment to be cloned is integrated for cloning
Recombinant viral vectors are genetic engineering tools commonly used for gene transfer purpose with high transfection efficiency and site specific gene insertion.
The document summarizes key information about Hepatitis C virus (HCV). It belongs to the Flaviviridae family of viruses which includes yellow fever, West Nile virus, and dengue virus. HCV is a small enveloped virus with a positive sense RNA genome approximately 9,600 nucleotides in length. The genome contains a single open reading frame flanked by untranslated regions, and codes for structural and non-structural proteins. HCV enters the liver cells and uses its RNA for both translation and transcription to replicate itself and cause infection through multiple steps.
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This document describes research into producing recombinant adeno-associated virus (rAAV) using serum-free suspension cultures of HEK 293 cells. The researchers investigated the effects of different DNA, polyethyleneimine (PEI) transfection reagent, and cell concentrations on rAAV yields. They found that transfecting at a cell concentration of 3x10^6 cells/mL resulted in a 2.5-3.5 fold increase in infectious virus particles (IVP) and genomic particles (Vg) compared to 1x10^6 cells/mL. When testing different PEI:DNA ratios, IVP ranged from 1.19x10^8 to 1.03x10^9 IVP/
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HCV was discovered in 1989 as a major cause of non-A, non-B hepatitis. It is a single stranded RNA virus of the Flaviviridae family. An estimated 130-170 million people worldwide are chronically infected, making it a significant global health burden.
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This document discusses chemiluminescence immunoassay (CLIA), a technique that uses chemiluminescence to label antibodies or antigens. There are four main types of CLIA: chemiluminescence immunoassay, chemiluminescence enzyme immunoassay, microparticle chemiluminescence immunoassay, and electrochemiluminescence immunoassay. CLIA offers advantages like high sensitivity, specificity, speed, and automation. However, it also has disadvantages such as requiring specialized equipment and potential lot-to-lot variability in reagents. The document provides details on CLIA procedures like the sandwich CLIA technique.
TEST BANK For Community Health Nursing A Canadian Perspective, 5th Edition by...Donc Test
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Histololgy of Female Reproductive System.pptxAyeshaZaid1
Dive into an in-depth exploration of the histological structure of female reproductive system with this comprehensive lecture. Presented by Dr. Ayesha Irfan, Assistant Professor of Anatomy, this presentation covers the Gross anatomy and functional histology of the female reproductive organs. Ideal for students, educators, and anyone interested in medical science, this lecture provides clear explanations, detailed diagrams, and valuable insights into female reproductive system. Enhance your knowledge and understanding of this essential aspect of human biology.
Integrating Ayurveda into Parkinson’s Management: A Holistic ApproachAyurveda ForAll
Explore the benefits of combining Ayurveda with conventional Parkinson's treatments. Learn how a holistic approach can manage symptoms, enhance well-being, and balance body energies. Discover the steps to safely integrate Ayurvedic practices into your Parkinson’s care plan, including expert guidance on diet, herbal remedies, and lifestyle modifications.
Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
Basavarajeeyam is an important text for ayurvedic physician belonging to andhra pradehs. It is a popular compendium in various parts of our country as well as in andhra pradesh. The content of the text was presented in sanskrit and telugu language (Bilingual). One of the most famous book in ayurvedic pharmaceutics and therapeutics. This book contains 25 chapters called as prakaranas. Many rasaoushadis were explained, pioneer of dhatu druti, nadi pareeksha, mutra pareeksha etc. Belongs to the period of 15-16 century. New diseases like upadamsha, phiranga rogas are explained.
Does Over-Masturbation Contribute to Chronic Prostatitis.pptxwalterHu5
In some case, your chronic prostatitis may be related to over-masturbation. Generally, natural medicine Diuretic and Anti-inflammatory Pill can help mee get a cure.
8 Surprising Reasons To Meditate 40 Minutes A Day That Can Change Your Life.pptxHolistified Wellness
We’re talking about Vedic Meditation, a form of meditation that has been around for at least 5,000 years. Back then, the people who lived in the Indus Valley, now known as India and Pakistan, practised meditation as a fundamental part of daily life. This knowledge that has given us yoga and Ayurveda, was known as Veda, hence the name Vedic. And though there are some written records, the practice has been passed down verbally from generation to generation.
Here is the updated list of Top Best Ayurvedic medicine for Gas and Indigestion and those are Gas-O-Go Syp for Dyspepsia | Lavizyme Syrup for Acidity | Yumzyme Hepatoprotective Capsules etc
- Video recording of this lecture in English language: https://youtu.be/kqbnxVAZs-0
- Video recording of this lecture in Arabic language: https://youtu.be/SINlygW1Mpc
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
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Osteoporosis - Definition , Evaluation and Management .pdfJim Jacob Roy
Osteoporosis is an increasing cause of morbidity among the elderly.
In this document , a brief outline of osteoporosis is given , including the risk factors of osteoporosis fractures , the indications for testing bone mineral density and the management of osteoporosis
Local Advanced Lung Cancer: Artificial Intelligence, Synergetics, Complex Sys...Oleg Kshivets
Overall life span (LS) was 1671.7±1721.6 days and cumulative 5YS reached 62.4%, 10 years – 50.4%, 20 years – 44.6%. 94 LCP lived more than 5 years without cancer (LS=2958.6±1723.6 days), 22 – more than 10 years (LS=5571±1841.8 days). 67 LCP died because of LC (LS=471.9±344 days). AT significantly improved 5YS (68% vs. 53.7%) (P=0.028 by log-rank test). Cox modeling displayed that 5YS of LCP significantly depended on: N0-N12, T3-4, blood cell circuit, cell ratio factors (ratio between cancer cells-CC and blood cells subpopulations), LC cell dynamics, recalcification time, heparin tolerance, prothrombin index, protein, AT, procedure type (P=0.000-0.031). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and N0-12 (rank=1), thrombocytes/CC (rank=2), segmented neutrophils/CC (3), eosinophils/CC (4), erythrocytes/CC (5), healthy cells/CC (6), lymphocytes/CC (7), stick neutrophils/CC (8), leucocytes/CC (9), monocytes/CC (10). Correct prediction of 5YS was 100% by neural networks computing (error=0.000; area under ROC curve=1.0).
2. 2
Adeno-associated virus (AAV)
Introduction of AAV
Adeno-associated virus (AAV) is a small single-strand DNA virus infecting human and some other primate species.
Currently, AAV has not known to cause disease and only induces very mild immune responses. As a member of the
family Parvoviridae, wild type AAV requires the assistance of adenovirus or herpesvirus to complete the duplication,
which is the reason why it’s called adeno-associated virus [1,2]. The wild-type AAV2 genome consists of the viral
rep and cap genes (encoding replication and capsid genes, respectively), flanked by inverted terminal repeats (ITRs)
that contain all the cis-acting elements necessary for replication and packaging. The genome of typical AAV2 is
about 4800bp, consisting of two upstream and downstream open read frames (ORFs) which are between two
inverted terminal repeats (ITR) comprising Rep and Cap. ITR is required for synthesis of complementary DNA
strand, while Rep and Cap can be translated into various proteins, including AAV virus cycle essential protein
Rep78, Rep68, Rep52, Rep40 and enveloped protein VP1, VP2, VP3, etc. [3].
The present recombinant AAV (rAAV) vectors are generated by replacing all of the viral genome between the ITRs
with a transcriptional cassette of less than 5 kilobases in length. The resulting construct is then co-expressed with
two other plasmids: 1) an AAV-RC plasmid that provides the Rep and Cap genes in trans (separate from the
ITR/Transgene cassette) and 2) an AAV helper plasmid that harbors the adenoviral helper genes. AAV-293 cells are
used as the packaging cell line since they provide the E1a protein in trans as well. By modifying the Rep and Cap
genes, scientists can control the serotypes to guide the recombinant AAV infection towards certain tissues.
This 3-plasmid co-transfection system liberates the need for adenovirus during AAV production, which greatly
simplifies the purification process.
To date, a total of 12 serotypes of AAV have been described with their unique traits and tropisms [4]. Concerning
high safety, low immunogenicity, long-term expression of exogenous genes, AAV is thought to be the best gene
delivery tool for gene function research in vivo.
Over the past decades, numerous AAV serotypes have been identified with variable tropism. To date, 12 AAV
serotypes and over 100 AAV variants have been isolated from adenovirus stocks or human/nonhuman primate
tissues. Different AAV serotypes exhibit different tropisms, infecting different cell types and tissue types. So,
selecting the suitable AAV serotype is critical for gene delivery to target cells or tissues.
Due to the exhibition of natural tropism towards certain cell or tissue types, rAAV has garnered considerable
attention. Highly prevalent in humans and other primates, several AAV serotypes have been isolated. AAV2, AAV3,
AAV5, AAV6 were discovered in human cells, while AAV1, AAV4, AAV7, AAV8, AAV9, AAV10, AAV11,
AAV12 in nonhuman primate samples [5,6]. Genome divergence among different serotypes is most concentrated on
hypervariable regions (HVRs) of virus capsid, which might determine their tissue tropisms. In addition to virus
capsid, tissue tropisms of AAV vectors are also influenced by cell surface receptors, cellular uptake, intracellular
processing, nuclear delivery of the vector genome, uncoating, and second-strand DNA conversion [7].
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3. 3
In order to better improve the infection efficiency and specificity of AAV to target tissues, scientists have genetically
modified the viral capsid, and generated mosaic vectors to create chimeric AAV by swapping domain’s or amino-
acids between serotypes [8,9], which may allow researchers to specifically target cells with certain serotypes to
effectively transduce and express genes in a localized area [10].
Protocol Overview
A schematic overview of recombinant AAV production is shown in Figure 1. The first step is to clone the gene of
interest (GOI) into an appropriate plasmid vector. For most applications, the cDNA of interest is cloned into one of
the ITR/MCS containing vectors. The inverted terminal repeat (ITR) sequences present in these vectors provide all
of the cis-acting elements necessary for AAV replication and packaging.
The recombinant expression plasmid is co-transfected into the AAV-293 cells with pHelper (carrying adenovirus-
derived genes) and pAAV-RC (carrying AAV2 replication and capsid genes), which together supply all of the trans-
acting factors required for AAV replication and packaging in the AAV-293 cells. Recombinant AAV viral particles
are prepared from infected AAV-293 cells and may then be used to infect a variety of mammalian cells.
Upon infection of the host cell, the single-stranded virus must be converted into double-stranded for gene expression.
The AAV virus relies on cellular replication factors for the synthesis of the complementary strand. This conversion
is a limiting step in recombinant gene expression and can be accelerated using adenovirus superinfection or
etoposides, such as camptothecin or sodium butyrate. Whereas these agents are toxic to the target cells and can kill
target cells if left on the cells, so the use of etoposides is only recommended for short-term use or in obtaining viral
titers. Typically, the AAV genome will form high molecular weight concatemers which are responsible for stable
gene expression in cells.
Figure 1. AAV packaging experiment flow chart.
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4. 4
Experimental Materials
GeneMedi's AAV Vectors System
GeneMedi's AAV Vector System, also named AAV expression system or AAV packaging plasmid system, is a
powerful tool for in-vivo gene delivery, gene editing, and gene therapy. You can easily produce a recombinant AAV
(rAAV) particle in 293T cell line in high titer using GeneMedi's AAV Vector System. The Genemedi AAV vector
system including multiple AAV expression vector plasmids, AAV helper plasmid and the serotypes-specific AAV
Rep-Cap plasmids (AAV-RC).
GeneMedi's AAV expression vectors have been inserted with different expression cassettes, containing kinds of
verified protomers and reporters including GFP, zsgreen, RFP, mcherry and luciferase. The GeneMedi's AAV
expression vectors have been proved very suitable for unique gene overexpression or shRNA-mediated knock-down
(also called RNAi (RNA interference ). You can also achieve gene knock-out(KO) or gene editing using our Crispr-
cas9-gRNA AAV expression vector.
The serotypes-specific AAV Rep-Cap plasmid (AAV-RC plasmid, or called AAV-RC plasmid) contain the AAV2-
Rep gene with different serotypes of AAV's Cap gene(also called AAV capsids gene).
GeneMedi's AAV Rep-Cap plasmids is including AAV2, AAV5, AAV6, AAV8, AAV9, AAV-PHP.B, AAV-
PHP.eB, AAV PHP.s, AAV-Retro (Retrograde), AAV-Anc80 (L65), AAV-DJ, AAV-DJ8. GeneMedi also supplies
capsid optimized AAV variant including AAV2 variant(Y444F), AAV2 variant (Y272F, Y444F, Y500F, Y730F),
AAV2 variant (Y444F, Y730F, Y500F, Y272F, Y704F, Y252F), AAV2.7m8, AAV8 variant (Y733F), AAV8
variant(Y733F, Y447F), AAV8 variant(Y733F,Y447F,Y275F) and some other engineering AAV serotypes not
mentioned.
Visit https://www.genemedi.net/i/aav-vector-system here for more information about GeneMedi’s AAV vector
system and multiple serotypes of AAV Rep-Cap plasmids.
Bacterium Strain
E. coli strain DH5ɑ is used for amplification of shuttle and backbone vectors.
Packaging Cell Line
AAV-293 is the virus packaging cell line that can facilitate initial production, amplification and titration of rAAV.
Originated from the 293 cell line and established for plaque assays, this cell line was identified to be an easy-to-
handle transfection host.
The complete growth medium of AAV-293 is Dulbecco's Modified Eagle Medium (DMEM) supplemented with
10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin (Pen-Strep). For a continuous culture, cells should
not exceed 70% confluence to maintain proper characteristics. Usually, starting from cell passage number one,
optimal results can be obtained within 30 passages. Once reached, it is best to start a new culture from another
frozen stock in case of any unexpected mutations and unhealthy growth. Therefore, banking your own AAV-293
frozen stocks is very important to ensure experimental integrity and continuity. Freezing cells at the logarithmic
phase will improve post-thaw viability.
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5. 5
Notices:
To maintain cells in a healthier condition and improve production efficiency of AAV, it is recommended to use our
Genemedi anti-mycoplasma reagent, CurePlasmaTM
.
Other Materials and Reagents
Gene of interest
LB broth
Agar and Agarose
Kanamycin
Ampicillin
70 and 100% ethanol
Sterile PBS
Iodixanol
Pluronic-F68
Chlorine bleach
DNA gel apparatus and power supplies
Class II Biosafety Cabinet
37℃ orbital shaker
37℃ bacteria incubator
37℃, 5% CO2 incubator
15- and 50-ml conical tubes
25- and 75-cm2
tissue culture flasks
Cell scrapers
Dry-ice/methanol bath
Liquid nitrogen tank
Low-speed swinging-bucket centrifuge
Microcentrifuge
Centrifuge tube (thick-wall polycarbonate tube with cap)
Packaging and Concentration of rAAV
Construction and Amplification of Plasmids
Constructed rAAV vector plasmids and the backbone vector should be amplified in DH5ɑ and purified by Maxi-
Prep kit to remove endotoxins (note: QIAGEN Plasmid Maxi Kit is recommended). A concentration over 1 µg/µl
and the A260/A280 ratio range between 1.7-1.8 is required for virus packaging. Please be cautious that plasmid
quality would affect the transfection efficiency and titer of product viruses.
Note:
In order to construct vectors quickly and efficiently, it is strongly recommended to use Genemedi - ClonEasyTM
One
Step Cloning Kit(Cat. GM-GC-01/02/03).
Click here to find more about Genemedi - ClonEasyTM
One Step Cloning Kit
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6. 6
Transfection of Virus Plasmids into AAV-293 Packaging Cells
a. AAV-293 cells should be prepared at least a day ahead to reach a confluence of 50%-70% monolayer
morphology before transfection.
b. On the day of transfection, DMEM needs to be pre-warmed at 37℃ water bath and LipoGeneTM
transfection
reagent should be equilibrated to room temperature and tapped to mix before use.
c. To prepare viral plasmids for each reaction using a 10-cm dish according to the following table 1:
Table 1. Plasmid and transfection reagent required for transfection.
Component Amount
pAAV-RC 10 μg
pHelper 20 μg
pAAV-GOI 10 μg
LipoGeneTM
100 l
d. Mix plasmids with transfection reagent in DMEM and add drop-wise to pre-seeded AAV-293 cells. Incubate in
37℃, 5% CO2 and refresh with complete culture medium in 6 hours.
Note:
1. A detailed protocol of the transfection reagent can be referred to Genemedi LipoGeneTM
Transfection Reagent
User Manual.
Click here to find more about Genemedi - LipoGeneTM
Transfection Reagent
2. Cells should be in a healthy growth state for use before transfection.
Collection of AAV
a. Around 72 hours after transfection, harvest the cells from the 10 cm plate with a cell scraper.
b. Spin the cells at 1,500g for 5 minutes to collect the cell pellet. Resuspend the cell pellet in 0.5ml lysis buffer (10
mM Tris-HCl (pH8.5), 150 mM NaCl). Freeze/thaw the cell pellet 3 times through a dry ice/ethanol bath and a
37°C water bath to obtain the crude lysate.
c. Spin down the crude lysate at 3,000g for 10 minutes. Collect the supernatant fraction, which contains harvested
rAAV. Keep the virus at -80℃.
Purification of AAV
a. Reagent Preparation:
1× PBS-MK buffer: Dissolve 52.6 mg of MgCl2, and 29.82 mg of KCl in 1× PBS in a final volume of 200 mL.
1 M NaCl/PBS-MK buffer: Dissolve 5.84 g of NaCl in 1× PBS-MK buffer in a final volume of 100 mL.
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7. 7
Note: The buffer should be sterilized by passing through a 0.22-μm filter and store at 4 ℃.
15% iodixanol: mix 4.5 mL of 60% iodixanol and 13.5 mL of 1 M NaCl/PBS-MK buffer;
25% iodixanol: mix 5 mL of 60% iodixanol and 7 mL of 1x PBS-MK buffer and 30 μL of phenol red;
40% iodixanol: mix 6.7 mL of 60% iodixanol and 3.3 mL of 1x PBS-MK buffer;
60% iodixanol: mix 10 mL of 60% iodixanol and 45 μL of phenol red.
b. Overlay each solution into a QuickSeal tube in the order (5 mL of 60% iodixanol; 5 mL of 40% iodixanol; 6 mL
of 25% iodixanol; 8 mL of 15% iodixanol) using a 10 mL syringe and an 18 g needle. Carefully add up to 5 mL
of clarified virus supernatant on top of the gradient. Use 1× PBS (or cell lysis buffer) to top off the tube. Seal
the QuickSeal tubes. Centrifuge at 350,000 g for 90 min in a T70i rotor at 10 ℃.
c. Carefully take the QuickSeal tubes out of the rotor and place them in a stable rack.
Note:
Take care to avoid bubbles during purification.
If more centrifugation time is needed, you can alternatively centrifuge for 2 h at 200,000 g at 18 ℃.
Make sure not to disturb the gradient before virus collection.
d. Pierce the QuickSeal tube slightly below the 60-40% interface with an 18 g needle attached to a 10 ml syringe.
The bevel of the needle should be up, facing the 40% iodixanol step. Collect up to 5 ml per tube, and avoid
collecting the proteinaceous material at the 40-25% interface.
Note:
Unless otherwise specified, all AAV viruses are purified with iodixanol gradient ultracentrifugation at 350,000g for
90 min in a T70i rotor at 10℃ to separate contaminants from the impure AAV preparations. The 15% iodixanol step
helps destabilize ionic interactions between macromolecules with the addition of 1M NaCl. The 40% and 25% steps
are used to remove contaminants with lower densities, including empty capsids, while the 60% step acts as a cushion
for genome-containing virions. After several steps of gradient ultracentrifugation, the virus particles will be
enriched.
Concentration of AAV (Ultrafiltration method)
It may be necessary to remove iodixanol (molecular weight: 1,550 Dalton) from the gradient fractions with filtration
or dialysis method either to concentrate the viral particles or avoid interfering with some add-on processes.
a. Pluronic-F68 Preparation:
0.1% Pluronic-F68: 49.5 ml PBS + 500 µl 10% Pluronic F68
0.01% Pluronic-F68: 45 ml PBS + 5 ml 0.1% Pluronic-F68
0.001% Pluronic-F68 with 200mM NaCl: 45 ml PBS + 5 ml 0.01% Pluronic-F68 + 200 mM NaCl
0.001% Pluronic-F68 (formulation buffer): 45 ml PBS + 5 ml 0.01% Pluronic-F68.
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8. 8
b. Balance the filter membrane with 15 ml of 0.1% Pluronic F68 and incubate for 10 min at room temperature.
c. Remove the 0.1% Pluronic F68 and add 15 ml of 0.01% Pluronic F68.
d. Centrifuge at 3000 rpm for 5 min at 4 ℃.
e. Discard the filtrates and add 15 ml of 0.001% Pluronic F68 with 200mM NaCl PBS.
f. Centrifuge at 3000 rpm for 5 min at 4 ℃.
g. Discard the filtrates and add virus purifications.
h. Centrifuge at 3500 rpm for 8 min at 4 ℃, discard the flow-through.
i. Replenish more virus purifications and spin 3500 rpm for 4 min at 4°C, discard the filtrates. Repeat this step as
needed.
j. Utilize a P1000 to pipette up and down to wash off the filter wall to recover as much virus as possible.
k. Keep the concentrates at 4 ℃ for short term (less than one week), or in small aliquots and keep at -80 ℃ for long
term usage.
Note:
1. It is difficult to remove Iodixanol. Add more formulation buffer and virus purifications and pipet back and forth
a few times in order to mix the iodixanol settled at the bottom or wall of the column into the solution after each
centrifugation.
2. Concentrating on a minimum of 500 µl is recommended. If the concentrate volume is less than 500 µl, bring up
the volume with formulation buffer.
Quality Assurance of rAAV
AAV capsid contains VR1 82kDa, VR2 72kDa and VR3 62kDa, which can be detected using the method of
polyacrylamide gel electrophoresis (PAGE) followed by silver staining or Coomassie blue staining. Pure AAV
should display only three major protein bands, such as the following virus purified in Genemedi shown in Figure 2.
We guarantee the purity of the AAV virus based on the specification that we give for different services.
Figure 2. Purity of purified AAV virus.
Titer Detection of rAAV
AAV titers are determined by real-time quantitative PCR using primers targeted the ITR. The amplicons are detected
using SYBR green technology. Titer values are then determined by comparison to a standard curve of a plasmid
sample of known concentration. An example of the Ct value of the standard sample and sample to be tested is shown
in table 2, while the standard curve is displayed in figure 3.
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Table 2. Ct value of standard and sample in a typical absolute quantification process.
Set the average Ct value of each group as Y-axis, the logarithm of corresponding group as X-axis. Substitute the
average Ct value of samples to be tested into formula, obtaining X = 7.45. Substitute the AAV virus titer calculation
formula: 10x
× 40000 vg/ml= 107.45
× 40000=1.1 × 1012
vg/ml.
Figure 3. Standard curve in absolute quantification.
Cell Infection Test of AAV
After AAV titer detection, the infection activity needs to be evaluated before animal experiments. Test the
expression of target gene by infecting cells, such as 293T, CHO. MOI will be controlled ranging from 104
to 105
(MOI is multiplicity of infection, namely the number of virus particles needed for infecting a cell). The detailed
AAV infection protocol can be found in the AAV User Manual.
Safe Use of AAV
1. AAV related experiments should be conducted in biosafety level 2 facilities (BL-2 level).
2. Please equip with lab coat, mask, gloves completely, and try your best to avoid exposing hand and arm.
3. Be careful of splashing virus suspension. If biosafety cabinet is contaminated with virus during operation, scrub
the table-board with solution comprising 70% alcohol and 1% SDS immediately. All tips, tubes, culture plates,
medium contacting virus must be soaked in chlorine-containing disinfectant before disposal.
4. If centrifuging is required, a centrifuge tube should be tightly sealed. Seal the tube with parafilm before
centrifuging if condition allowed.
Standard Copy Number (vg/ml) Ct value 1 Ct value 2 Ct value 3
Standard_1 1010
4.51 4.32 4.42
Standard_2 109
7.21 7.61 7.25
Standard_3 108
10.97 10.55 10.67
Standard_4 107
14.19 14.38 14.35
Standard_5 106
17.75 17.58 17.68
AAV Sample 12.78 12.65 12.81
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5. AAV related animal experiments should also be conducted in BL-2 level.
6. AAV associated waste materials need to be specially collected and autoclaved before disposal.
7. Wash hands with sanitizer after experiment.
Storage and Dilution of AAV
Storage of AAV
Virus can be stored at 4°C for a short time (less than a week) before using after reception. Since AAV viruses are
sensitive to freeze-thawing and the titer drops with repeated freeze-thawing, aliquot viral stock should be stored at -
80°C freezer immediately upon arrival for long-term usage. While virus titer redetection is suggested before using if
the AAV viruses have been stored for more than 12 months.
Dilution of AAV
Dissolve virus in ice water if virus dilution is required. After dissolving, mix the virus with medium, sterile PBS or
normal saline solution, keeping at 4°C (using within a week).
Precautions
· Avoid AAV exposure to environmental extremes (pH, chelating agents like EDTA, temperature, organic solvents,
protein denaturants, strong detergents, etc.)
· Avoid introducing air into the AAV samples during vortex, blowing bubbles or similar operations, which may
result in protein denaturation.
· Avoid repeated freezing and thawing.
· Avoid exposing to “regular” plastics (especially polystyrene or hydrophobic plastics) for prolonged periods in
liquid phase. Most AAV viruses are very sticky and loss can occur if exposed to regular plastics, including tubes,
cell culture plates, pipette tips, if not frozen. It is best to store AAV in siliconized or low protein binding tubes.
Pluronic F-68 used at 0.01%-0.1% in the formulation buffer will minimize sticking if regular plastics are used.
· Avoid diluting AAV into low salt solution. Some AAV serotypes, such as AAV2, aggregates in low salt solution,
which will be non-infectious.
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11. 11
References
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