Q: What is the maximum size of an rAAV transgene?
A: The upper limit of rAAV genome packaging is ~ 5Kb including the required 145bp ITR sequences at either end. Thus, rAAV accommodate a ~4.5Kb transgene expression cassette. An rAAV transgene expression cassette usually includes a promoter, a gene of interest, and a terminator signal. PackGene’s K104 vector has been designed to maximize gene of interest capacity by integrating the smallest available mammalian promoter region in miniCMV (180 bp) and terminator region (50 bp). PackGene’s K104 vector can thus accept a gene of interest up to 4.4kb in length.
Q: What are rAAV serotypes, and what steps should be taken to ensure that the best serotype is chosen?
A: Different AAV serotypes are defined by differences in the amino acid sequence and three-dimensional structure of their capsid proteins, and more than 200 AAV serotypes of have been discovered or designed. Serotype specific differences in rAAV capsid proteins correspond with variations in cell surface receptor recognition and binding. This, in turn, results in variations in the infection rate of rAAV serotypes across tissues and cell types.
PackGene’s expert technical team is available to help you determine the optimum serotype for your experiments based on the literature regarding rAAV serotype infection rates and our own internal testing. Nevertheless, for target cell types or tissues without substantial literature available, we may recommended the execution of pilot experiments using reporter transgenes to determine the most ideal serotype.
Q: What information will I need to provide to place a custom rAAV vector construction order?
A: Custom AAV vector construction projects can be generated in several ways, and our expert technical team is available to help in the design process. There are several questions that you may prepare answers for to expedite the design process, they are:
- Do you have a transgene template?
If so, please provide your gene template to us for verification. - Do you want to overexpress a gene without a template?
If so, please provide the gene number, sequence map, host species, and gene length. - Do you want to generate rAAVs for the manipulation of gene expression using techniques such as RNAi or CRISPR?
If so please provide us with the target gene number, host species, and gene length. - Is there a specific promoter sequence you would like to use?
- Is there a specific fluorescent tag or reporter that you would like to use?
- Will several transgenes need to be co-expressed simultaneously?
- Is your total transgene sequence length <4.4kb?
Q: What information will I need to provide to place a custom rAAV packaging order?
A: Custom AAV packaging projects can be generated in several ways, and our expert technical team is available to help in the design process. There are several questions that you may prepare answers for to expedite the design process, they are:
- Do you know which serotype you would like you use for your project?
Our expert team is available to help guide your selection if you would like, but you may alternatively find your ideal serotype by looking toward the literature within your field. - Are you confident that your preferred serotype is capable of infecting the cells that you plan to use for your experiments?
If not, we offer fluorescent control test kits for screening various serotypes. These can be used to define the infectivity of a given serotype in your cells, or to determine the optimal serotype for your experiments. - Do you plan to provide your own plasmid for packaging?
If so, please make sure that you to provide a vector map and full sequence. Additionally, it is best to confirm ITR spacing and to make sure that the plasmid has been fully sequenced to avoid complications associated with common mutations that can be driven by the presence of ITRs. - What are your requirements for the amount, titer, and packaging of the final deliverable rAAV?
Q: How much plasmid do I need to provide for AAV packaging?
A: You only need to provide 1-4 µg of plasmid. We will handle the plasmid preparation necessary for AAV packaging. You don’t need to purchase an additional plasmid prep service unless you wish to receive more plasmid from us. Please note, the timeline in our quote already includes the plasmid preparation.
Q: What are the difference between research and NHP grade?
Q: How do you choose the fluorescent or luminescent marker for live imaging in mice?
A: For in vivo imaging, it’s generally advised to use vectors with luciferase.
Currently, in vivo imaging primarily utilizes two techniques: bioluminescence and fluorescence. Bioluminescence involves using the luciferase gene to label cells or DNA, while fluorescence employs fluorescent proteins such as GFP, EGFP, RFP, YFP, mCherry, etc., to mark cells or proteins. Bioluminescence offers advantages like straightforward operation, sensitive response, rapid imaging, and clear visualization. However, its drawback lies in its relatively weak signal, necessitating the use of CCD lenses for detection and requiring instruments with high precision. On the contrary, fluorescence allows for the utilization of various proteins for labeling and enables multiplex labeling, making the process relatively straightforward. Nevertheless, nonspecific fluorescence imposes limitations on its sensitivity, necessitating the use of excitation lights of different wavelengths, thereby making precise in vivo quantification challenging. Bioluminescence relies on the interaction with luciferase to emit light, demonstrating high specificity. The red light emitted by luciferase penetrates tissues nearly 100 times more effectively in vivo than the green light emitted by green fluorescent protein, resulting in a higher signal-to-noise ratio. While fluorescent proteins necessitate excitation light to produce reflected light, nonspecific fluorescence from the mouse’s fur reduces the signal-to-noise ratio during the detection process. Fluorescent protein detection is more suited to ex vivo detection, whereas luciferase detection is better suited to in vivo detection. Currently, luciferase labeling is more commonly employed. There are two frequently used luciferases: Firefly Luciferase (Fluc) and Renilla Luciferase (Rluc), each utilizing different substrates—D-Luciferin for the former and Coelenterazine for the latter. They emit light of varying colors, with the former emitting light at approximately 560nm and the latter emitting light at approximately 450-480nm. The light emitted by the former penetrates tissues more effectively, while the latter undergoes faster metabolism in vivo compared to the former. Typically, the former is utilized as a reporter gene, although both can be simultaneously employed for dual labeling.
Q: What quality control tests do you conduct for your AAV?
A: Our AAV products are subjected to standard release testing procedures, including endotoxin assessment using Limulus Amebocyte Lysate (LAL) assay, purity analysis via Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), and titer determination using quantitative Polymerase Chain Reaction (qPCR) or droplet digital PCR(ddPCR). Moreover, we conduct restriction enzyme digestion for the Gene of Interest (GOI) plasmids utilized in packaging. Different grade AAV may include different QC tests as listed here.
In addition, we offer 40+ analytical tests to measure titer, AAV genome integrity, characterization, purity, aggregation, contamination and safty, including TEM, AUC, TCID50, Nanopore deep sequencing and may others. Please refer to our anlytical tests webpage.
Q: How is the titer of AAV determined?
A: During AAV titer measurement, our instruments are initially calibrated using the AAV standard product ATCC VR-1816™, a globally recognized reference titer verified by 16 laboratories. Subsequently, we employ SYBR Green qPCR methodology to ascertain titers, achieving values of 1E+13GC/ml or higher. This meticulous approach ensures alignment with prevailing academic standards and prevents inaccurate titration results.
Besides qPCR, we have several other methods available for AAV titer determination:
- Genome titer detection by ddPCR.
- Capsid titer detection by ELISA technology.
- Infectious titer detection by TCID50.
Q: What is the maximum exogenous gene capacity in rAAV as a gene delivery vector?
Q: What are the difference between scAAV and ssAAV?
Q: What does GC/ml means in rAAV titer, and how is it quantified?
Q: What methods are employed for AAV purification, and what QC tests are included?
A: PackGene produces high-purity AAV, purified using iodixanol density gradient centrifugation and multiple steps of filtration and sterilization.
PackGene’s offers QC tests for our AAV product depend on the AAV-grade you choose, the detailed QC test comparison are listed here
Here are some QC tests we offer:
① Purity: Assessed by running SDS-PAGE followed by Coomassie blue stainig (research grade) or silver staining (NHP-grade) to observe purity.
② Titer: For research-grade or HT-grade AAV, PackGene calibrates using ATCC standard, then employs SYBR Green QPCR to detect titer, achieving a titer of 1E+13GC/ml or higher.For NHP-grade, we determing the titer via ddPCR with primers specifically complement to 5′ and 3′ ITR region.
③ Endotoxin detection: Endotoxin content does not exceed 10 EU/ml for research-grade and 1EU/ml for NHP-grade.You may request more stringent endotoxin threshold with additional fees.
④ TEM electron microscopy (optional): Observation of empty/full ratio through electron microscopy, with a rate below 30% for most common serotypes in research-grade AAV, or below 20% for most common serotypes in NHP-grade AAV.
⑤ HPLC based purity assessment (optional): We offer AEC-HPLC (Anion Exchange Chromatography – High Performance Liquid Chromatography) and SEC-HPLC(Size Exclusion Chromatography – High Performance Liquid Chromatography). Both methods are used for measuring the purity of Adeno-Associated Virus (AAV) preparations.
AEC-HPLC is particularly useful for separating and measuring AAV from impurities such as host cell proteins and nucleic acids, which may have a different net charge compared to the AAV particles.
SEC-HPLC separates molecules based on their size. SEC-HPLC is valuable for assessing the size distribution of AAV particles and separating AAV from aggregates, fragmented particles, or other macromolecules present in the sample.
⑥ Mass spectrometry analysis (optional): Determination of AAV serotype. We offer AAV Capsid Peptide Mapping test by HPLC-MS/MS, and Capsid Protein Molecular Weight and Ratio test by RP-HPLC-MS. AAV Capsid Peptide Mapping by HPLC-MS/MS involves analyzing the peptides within the capsid proteins of the Adeno-Associated Virus (AAV) using High-Performance Liquid Chromatography coupled with Tandem Mass Spectrometry (HPLC-MS/MS). Capsid Protein Molecular Weight and Ratio by RP-HPLC-MS” involves determining the molecular weight and ratio of the capsid proteins of the AAV using Reversed-Phase High-Performance Liquid Chromatography coupled with Mass Spectrometry (RP-HPLC-MS).
⑦ ddPCR titer detection (optional): compared to qPCR titer measurement, ddPCR offers high sensitivity, accuracy, and reproducibility.
For more additional QC test we provide, please check our AAV analytical serivce page.
Q: Do you perform any AAV QC tests to assess empty capsid rates? If so, what methodologies are used? What level of empty capsid rate do you typically achieve?
A: Typically, methods such as AUC, TEM, CyroTEM, or VG TITER/CAPSID TITER are employed. However, AUC, TEM, and CyroTEM are not suitable for QC release; they are better suited for quality research and process development. Currently, anion exchange chromatography HPLC is utilized for method development. We at PackGene can provide analysis method development services and validate them against CyroTEM and AUC test results.
Empty capsid rates vary for each serotype obtained through column chromatography.For research-grade and NHP-grade, we guarantee <30% or <20% empaty capsid rate by TEM for most common serotypes. For GMP production, we will test the empty capsid rate during procss development. For example, PackGene’s AAV9 production can be optimized through process improvements to maintain levels below 10%.
Q: Which serotypes does rAAV encompass, and how do you determine the suitable serotype?
A: As of now, nine naturally occuring serotypes of human AAV have been discovered (AAV1/2/3/4/5/6/7/8/9) and widely applied in scientific research. AAV10 and AAV11 were first discovered in non-human primates in 2004, and no cross-reactivity was observed between AAV10, AAV11, and AAV2, making them promising candidate vectors. Subsequently, researchers isolated AAV12 and AAV13 from simian adenovirus, with limited research on these serotypes currently. Based on these wild-type AAVs, researchers have developed many AAV mutants, such as AAV-DJ and the PHP series, through various modification strategies.
Due to differences in the spatial structure of capsid proteins among AAV serotypes, there are significant variations in their recognition and binding to cell surface receptors, leading to tropism of different AAV serotypes for different tissues. When selecting serotypes, experimental purposes can refer to AAV serotypes used in peer-reviewed literature. For example, AAV1 and AAV9 are more commonly used in brain research than other wild-type AAV serotypes, while AAV6 exhibits higher lymphocyte selectivity.
There are also many engineered serotypes that have been modified or engineered to enhance specific properties for gene therapy applications. These modifications can include alterations to the capsid proteins to change tissue tropism, improve transduction efficiency, evade immune responses, or increase payload capacity. Engineered AAV serotypes have been developed through various strategies such as directed evolution, rational design, or hybridization of existing serotypes. These engineered serotypes offer enhanced performance and versatility, making them valuable tools for targeted gene delivery in biomedical research and therapeutic applications.
PackGene offers nearly 100 serotypes for our packaging service to assist your research work.
Additionally, the development of AAV mutant serotypes with more tissue specificity and stronger infectivity is crucial for innovation in AAV-mediated gene delivery. PackGene provides comprehensive AAV serotype engineeringg services to offer you a one-stop solution.
However, despite the tissue tropism of wild-type AAVs to some extent, the infection of non-target tissues cannot be completely avoided. In such cases, combining tissue- or cell-specific promoters with serotypes can greatly enhance AAV specificity. PackGene offers various tissue-specific promoters, such as the muscle-specific promoter MHCK7-2 and the liver-specific promoter TBG669. Our piVector Design embed in our online ordering system offers various promoters including universal and tissue specific promoters. You may easily build your vector into our AAV backbones that have been rigorously verified for effective viral packaging.
Q: What features does AAV have comparing to other viral vectors?
A: AAV vectors stand out for their safety, low immunogenicity, ability to transduce non-dividing cells, and potential for long-term gene expression without integrating into the host genome. These features make them particularly attractive for gene therapy applications targeting diseases where long-term expression and safety are paramount.
However, the limited packaging capacity is a constraint when delivering larger genes. In contrast, vectors like adenovirus and HSV can carry larger genetic payloads but come with higher immunogenicity and safety concerns. Lentiviral and retroviral vectors offer stable, long-term expression through genome integration but carry risks associated with insertional mutagenesis.
By leveraging the unique advantages of AAV, such as tissue-specific targeting through various serotypes and a favorable safety profile, therapies can be designed for a range of genetic disorders with minimized risks. These characteristics contribute to the growing preference for AAV vectors in both research and clinical gene therapy programs.
Q: Is there a universal AAV in vivo delivery protocol?
Q: What are the general considerations when designing AAV iexperiment?
A: Serotype selection: If you are unsure which AAV serotype is most suitable for your experiments, we advise that you test the infection rates of 3 or more different serotypes in your experimental system with our rAAV fluorescent reporter constructs.
Gradient dilution infection: The level of transgene expression driven by rAAV may vary substantially across different genes. We therefore recommend that you perform 3-4 AAV gradient dose injections to determine the ideal gene expression level for each rAAV before performing any formal experiments.
Experimental control: We advise the use of a GFP positive control vector of the same serotype and promoter as your experimental vector.
Q: Any recommendations for AAV in vitro infection experiement?
A: Serotype selection: For cells cultured in vitro, AAV-DJ and AAV6 are the most common choices. In conventional cell culture, AAV-DJ can infect more than 80% of cells, while AAV6 has the strongest infective potency against blood-derived cells.
Gradient dilution infection: The level of transgene expression driven by rAAV may vary substantially across different genes. We therefore recommend that you perform 3-4 AAV gradient dose injections to determine the ideal gene expression level for each rAAV before performing any formal experiments and to verify that your experimental dose is not cytotoxic.
Assay time: usually 2-7 days after infection.
Q: How should I approach AAV in vivo delivery and experiment design?
Q: What are your recommendations for pre-experimental design?
A: Serotype selection: If you’re uncertain about the best AAV serotype for your experiments, we recommend testing at least three different serotypes in your system using our control rAAV with fluorescent reporter constructs.
Gradient dilution infection: Since transgene expression levels can vary by gene, we suggest performing 3-4 AAV gradient dose injections to identify the ideal expression level before starting formal experiments.
Experimental control: Use a GFP-positive control vector that matches the serotype and promoter of your experimental vector.
Q: What should I consider when using AAV for in vitro infection?
A: Serotype selection: For in vitro studies, AAV-DJ and AAV6 are common choices. AAV-DJ generally infects over 80% of cultured cells, while AAV6 is particularly effective with blood-derived cells.
Gradient dilution infection: Similar to in vivo experiments, we advise conducting 3-4 AAV gradient dose infections to find the optimal transgene expression level and to ensure the dose is not cytotoxic.
Assay time: Typically, assays should be performed 2-7 days post-infection to assess the effects accurately.
Q: What is the length range for PackGene’s mRNA production?
Q: Do you guarantee a specific yield of mRNA?
A: Yes, we guarantee the delivery of specific mRNA quantities as per the agreed specifications.
Q: What is the concentration of your mRNA?
Q: What buffer is used for mRNA?
Q: How should I store my mRNA?
Q: How should I store and handle mRNA in LNP?
Q: What is the concentration of mRNA in LNP?
Q: How does PackGene purify mRNA?
Q: What method does PackGene use for dsRNA removal?
Q: How does PackGene measure residual dsRNA?
A: Residual double-stranded RNA (dsRNA) can form during the in vitro transcription process used to synthesize mRNA, potentially triggering unwanted immune responses in humans. To detect and quantify trace amounts of dsRNA contaminants, PackGene uses a double-antibody sandwich ELISA.
The process starts with coating the wells of an ELISA plate with a monoclonal or polyclonal antibody specific to dsRNA. When the mRNA sample is added, any dsRNA present binds to the capture antibody. After incubation, unbound material is washed away, and a second, labeled antibody—also specific to dsRNA—is introduced. This second antibody attaches to the bound dsRNA, forming a “sandwich” complex.
The detection antibody is typically linked to an enzyme, such as horseradish peroxidase (HRP), which reacts with a substrate to produce a measurable signal (colorimetric, fluorescent, or luminescent). The intensity of the signal is proportional to the dsRNA concentration and is quantified using a microplate reader, with values compared to a standard curve of known dsRNA concentrations. This highly specific and sensitive method ensures the safety and purity of mRNA therapeutics by detecting low levels of dsRNA contamination.
Q: How is mRNA purity defined?
Q: What is the function of N1-methyl-pseudouridine (m1Ψ) in mRNA?
A: N1-methyl-pseudouridine (m1Ψ) is a modified nucleoside that plays several key roles when incorporated into mRNA:
- Enhanced Stability: mRNA with m1Ψ modifications demonstrates increased stability, prolonging the half-life and allowing for longer-lasting protein expression.
- Reduced Immunogenicity: m1Ψ reduces the immune response typically triggered by unmodified mRNA, preventing inflammation and degradation, making it more efficient for therapeutic delivery.
- Improved Translation Efficiency: m1Ψ enhances translation efficiency by promoting ribosome binding and the initiation of protein synthesis, resulting in higher protein output.
- Resistance to RNA Editing Enzymes: m1Ψ provides protection against RNA editing enzymes like ADAR, which can alter the mRNA sequence by editing adenosine residues. This ensures the integrity and fidelity of the mRNA sequence during protein synthesis.
Q: What modified bases does PackGene offer?
Q: What’s the function of capping?
A: The 5′ cap of mRNA is essential for several key processes in gene expression and stability:
- Translation Initiation: The 5′ cap, typically a modified guanosine nucleotide (m7G), is recognized by translation initiation factors, facilitating ribosome binding and the start of translation.
- Protection from Degradation: The cap shields the mRNA from exonucleases, which degrade RNA from the ends, thereby enhancing the molecule’s stability and lifespan.
- Nuclear Export: The cap aids in exporting mRNA from the nucleus to the cytoplasm, where translation occurs, by interacting with nuclear export factors.
- Translation Efficiency: It promotes efficient translation by ensuring correct ribosome positioning at the start codon, boosting the accuracy of protein synthesis.
- mRNA Processing Regulation: The cap also influences mRNA maturation, affecting processes like splicing and polyadenylation, and can impact alternative splicing patterns.
Q: What 5’ cap does PackGene offer?
Q: How does PackGene measure capping efficiency?
A: We assess capping efficiency using LC-MS. Although not part of our standard release QC test, we typically achieve >97% capping efficiency based on internal testing. Detailed information about the capping efficiency measurement method is available here.
Q: Does PackGene provide Cap Efficiency analysis as stand-alone service?
Q: Can I use my own UTRs for mRNA production at PackGene?
Q: What is the sequence requirement for the T7 promoter?
Q: Does PackGene provide mRNA codon optimization?
Q: Can I use plasmids from previous vector construction orders for mRNA production?
Q: What method does PackGene use for the poly(A) length assay?
Q: Can PackGene perform quality testing on customer-supplied mRNA?
A: Yes, we can perform quality testing on mRNA provided by customers. Please refer to the mRNA QC tests we offer here.
Q: How much mRNA is needed to transfect cell cultures?
Q: How much mRNA in LNP is needed for mouse injections?
Q: Does PackGene provide GMP-grade mRNA?
Q: What template does PackGene use for mRNA manufacturing, and how is it produced?
Q: Can PackGene co-encapsulate both gene-editing mRNA and sgRNA in LNP? Which LNP formulation should be used?
Q: What are the differences between in vitro grade and in vivo grade lentivirus?
Q: How much plasmid do I need to provide for lentivirus packaging?
Q: What QC tests do you conduct on your lentivirus?
A: Lentivirus quality control (QC) encompasses various tests to ensure the integrity and safety of lentiviral vectors for research and therapeutic applications. Our release QC testing primarily focuses on post-transduction titer, which provides a true functional titer(infectious titer) to prevent overestimation.
Post-transduction qPCR involves infecting cells with lentivirus followed by quantifying viral titers using quantitative PCR (qPCR), providing essential information about lentiviral transduction efficiency.
Transduction tests are performed by infecting cells with lentivirus and diluting them to count fluorescent cells. Bright field and fluorescent microscopy images are then analyzed to evaluate transduction efficiency.
Additional QC tests include p24 ELISA to measure the core capsid protein of HIV for accurate lentivirus titration, PCR-based mycoplasma testing to ensure absence of contamination, bioburden testing to quantify live microorganisms, and endotoxin testing using the Limulus amebocyte lysate (LAL) assay to confirm absence of endotoxin contamination.
These comprehensive QC tests ensure that lentiviral vectors are of high quality, free from contaminants, and suitable for downstream research and therapeutic applications.
Q: Which packaging system do you utilize for lentivirus production?
Q: What are the benefits of using the 3rd generation lentivirus system?
Q: How should lentivirus be stored, and what is their shelf life?
Q: What is the genome capacity between the 5′ LTR and 3′ LTR of lentivirus?
Q: What is the titer of your lentivirus?
Q: How is the lentivirus titer determined?
A: In contrast to the common practice among most vendors, who typically measure lentivirus titer directly using methods such as qPCR or p24 ELISA. The Traditional p24 ELISA kit is the most commonly published method for measuring lentiviral titer. The method is suitable for tittering native or purified recombinant virus. However, in crude (unpurified) lentiviral supernatant, significant concentrations of overexpressed p24 protein may be present that are not assembled into viral particles. This causes an extreme overestimation of lentiviral titer.
We employ a different approach. Our titer measurement focuses on the post-transduction titer determined by qPCR, which helps to eliminate any concerns regarding overestimation, providing infectious titers that are significantly higher (100-1000 times) than the physical titer measured by p24, ensuring greater accuracy and consistency for experimental applications.
Additionally, if the lentivirus carries a fluorescent protein, we can further validate the post-transduction titer by examining bright-field and fluorescent microscopy images obtained from serial dilutions of the virus.
Q: Is it possible to adjust the lentivirus titer according to specific requirements?
Q: What is the difference between physical titer and infectious titer?
Q: What is the buffer composition of the lentivirus?
Q: Which cell line is employed for lentivirus production?
Q: What are the disposal procedures for lentivirus and equipment or supplies that have been in contact with lentivirus?
Q: How much plasmid material is required for lentivirus packaging?
Q: Do you offer assistance design lentivirus plasmid?
Q: What is a plasmid?
Q: What are the components of plasmid vectors?
Q: What is the origin of replication (ori)?
A: An Origin of Replication (Ori) is a key DNA sequence in plasmids that signals the start of DNA replication within a host cell. It enables the plasmid to replicate independently. Selecting the right Ori is critical to ensure optimal plasmid functionality and stability. Here’s how to choose the appropriate Ori:
- Understand Your Host Organism: Determine the organism (bacteria, yeast, or mammalian cells) where the plasmid will be expressed. Different organisms have distinct replication systems, so your Ori must be compatible with the host’s machinery.
- Consider Copy Number: Ori affects plasmid copy number, which influences gene expression levels. High-copy plasmids are ideal for high gene expression, while low-copy plasmids provide stability and controlled expression. Other factors like promoter choice and growth conditions also affect protein expression.
- Evaluate Regulatory Elements: Some Ori regions contain regulatory sequences that control replication efficiency and stability. If you need precise control over replication timing, choose an Ori with the necessary regulatory features.
- Test and Optimize: It may be necessary to try different Ori sequences in pilot experiments to find the one that works best for your plasmid’s stability, replication, and gene expression.
Q: What is a promoter?
A: A promoter is a DNA sequence that acts as a molecular switch, controlling when and how genes are expressed. Positioned upstream of a gene, the promoter dictates the transcription of DNA into mRNA, which is then translated into proteins. Choosing the right promoter is essential for plasmid design.
Types of Promoters:
- Constitutive Promoters: These promote continuous gene expression without regulation. Examples include the lac promoter (E. coli) and the CMV promoter (mammalian cells).
- Inducible Promoters: These allow controlled gene expression by external inducers like chemicals or temperature changes. For instance, the lac promoter can be induced with IPTG.
- Repressible Promoters: Gene expression can be reduced under specific conditions. The trp promoter (E. coli) is repressible by tryptophan.
- Ubiquitous Promoters: These promote gene expression across a wide range of cells or tissues, commonly used in gene therapy.
- Tissue-Specific Promoters: These restrict gene expression to specific cell types, useful in developmental biology and gene therapy.
Promoter Selection Considerations:
- Expression Strength: Promoters differ in their ability to drive gene expression.
- Host Organism: The promoter must be compatible with the host organism’s RNA polymerase system.
- Regulation: Choose between constitutive or inducible promoters based on whether you want continuous or controlled expression.
- RNA Polymerase Specificity: Different RNA polymerases (I, II, III) in eukaryotic cells recognize specific promoters, influencing gene transcription.
- RNA polymerase I (RNA Pol I): Responsible for ribosomal RNA (rRNA) transcription.
- RNA polymerase II (RNA Pol II): Transcribes eukaryotic protein-coding genes.
- RNA polymerase III (RNA Pol III): Handles the transcription of small RNAs.
Q: What are commonly used eukaryotic promoters?
A: CMV (Cytomegalovirus): Known for strong expression in mammalian cells but may lead to cytotoxicity and gene silencing.
SV40 (Simian Virus 40): Provides moderate to strong expression in mammalian cells, though promoter silencing can occur over time.
EF-1α (Elongation Factor-1 alpha): Ensures strong, stable expression in mammalian cells, often used in stem cell research.
Tet-On/Tet-Off: Allows inducible expression in mammalian cells using tetracycline, offering precise control over gene activity.
U6: Commonly used for expressing small RNAs like shRNA or siRNA for gene knockdown studies.
PGK: Suitable for expressing transgenes in yeast and mammalian systems.
UAS (Yeast GAL1): Controlled by the GAL4 transcription factor, used for regulated gene expression in yeast.
To access and learn more promoters, please go to piVector Designer Gene elements library.
Q: What are reporter genes and tags?
A: Reporter Genes: These encode detectable proteins or enzymes, used to track gene expression or specific cellular conditions.
Fluorescent Proteins (e.g., GFP): Emit fluorescence under specific light wavelengths, allowing real-time visualization of protein localization.
Luciferase: Produces light during chemical reactions, useful for studying gene expression and signaling pathways.
Beta-Galactosidase: Converts substrates into colored products, used for visualizing gene expression as blue stains in cells.
Reporter Tags: Small protein sequences genetically fused to target proteins, helping in purification or detection.
Myc Tag: An 11-amino acid tag used in various assays for protein detection.
FLAG Tag: An 8-amino acid tag useful for purification and detection. It can be removed by specific enzymes if needed.
HA Tag: A 9-amino acid sequence often used for detecting and purifying proteins.
GST Tag: Enhances solubility and aids in protein purification, widely used in prokaryotic systems.
His Tag: Composed of six histidine residues, it facilitates easy purification of recombinant proteins through metal affinity chromatography.
Q: What is a multiple cloning site (MCS)?
A: The Multiple Cloning Site (MCS), also known as a polylinker, is a short segment of DNA that contains several unique restriction enzyme recognition sites. These sites allow researchers to insert a gene of interest into the plasmid at a specific location. The MCS simplifies the process of cloning because it offers flexibility in choosing which restriction enzymes to use, making it easier to incorporate foreign DNA fragments into the plasmid without disrupting other essential elements.
Today, seamless cloning methods are often preferred over using restriction enzymes in the MCS for gene insertion. However, the restriction enzyme sites within the MCS still provide a convenient location for plasmid linearization during seamless cloning.
Q: What is an open reading frame (ORF)?
Q: What are regulatory elements?
Q: What is a poly A signal?
Q: What is an antibiotic resistance gene?
Q: What bacteria strains do you use for cloning and plasmid preparation?
A: We use the strains below depend on different applications:
1. DH5α
Genotype: F–, φ80dlacZΔM15, Δ(lacZYA-argF)U169, recA1, endA1, hsdR17(rK–, mK+), phoA, supE44, λ–, thi-1, gyrA96, relA1
Applications: Commonly used for general cloning purposes and blue/white screening. Its mutations in recA and endA enhance plasmid stability and transformation efficiency, making it a go-to strain for many cloning applications.
2. Top10
Genotype: F–, mcrA, Δ(mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, recA1, araD139, Δ(ara-leu)7697, galU, galK, λ–, rpsL(StrR), endA1, nupG
Applications: Suitable for general cloning and blue/white screening with high transformation efficiency. It is often preferred when maximizing the number of transformants is critical.
3. Stbl3
Genotype: F–, mcrB, mrr, hsdS20(rB–, mB–), recA13, supE44, ara-14, galK2, lacY1, proA2, rpsL20(StrR), xyl-5, λ–, leu, mtl-1
Applications: Ideal for cloning vectors with repetitive elements, such as long terminal repeats (LTRs) in lentiviral plasmids. The recA mutation minimizes recombination, enhancing the stability of complex constructs.
4. XL-10
Genotype: TetR Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F´ proAB lacIqZΔM15 Tn10 (TetR) Amy CamR]
Applications: Optimized for high-efficiency transformation, particularly for large or methylated DNA constructs. It is also suitable for blue/white screening and for cloning unstable or toxic sequences due to the recA mutation.
5. NEB Stable
Genotype: F´ proA+B+ lacIq Δ(lacZ)M15 Tn10 (TetR) endA1 recA1 hsdR17(rK– mK+) glnV44 λ– thi-1 gyrA96 relA1 spoT1
Applications: Designed to maintain unstable plasmids that might recombine or degrade in other strains. It is ideal for cloning large or recombination-prone plasmids, offering good yield and stability for long-term propagation.
Q: How is a plasmid constructed based on your design?
A: We use seamless cloning to construct most plasmids.
Seamless cloning is a method that allows precise insertion of DNA fragments into plasmid vectors without adding extra nucleotides at the junctions, a common issue with traditional restriction enzyme methods. This is crucial for protein expression, as even small additional amino acids can affect protein function. There are various approaches to seamless cloning, including techniques like overlap extension PCR or commercial kits.
Key Steps in Seamless Cloning:
Creation of Overlapping Ends: The DNA fragments are generated through PCR with ends that overlap with each other or the plasmid. These overlaps are designed into the primers used for amplification.
Annealing of Overlapping Ends: The complementary overlapping regions hybridize when mixed together.
Extension and Ligation:
A polymerase may extend the annealed fragments, filling in gaps.
DNA ligase then seals the nicks, or a commercial system may combine both enzymatic steps in a single process.
Requirements:
DNA Fragments with Overlaps: These fragments, usually created by PCR, must have complementary sequences for hybridization.
Polymerase: A polymerase is needed for filling in gaps, if required.
DNA Ligase: Ligase seals the nicks unless using a system that combines all steps.
Pros and Cons:
Pros:
Enables precise, in-frame gene insertions without unwanted nucleotides.
No need for restriction enzyme sites, giving more design flexibility.
Simplified primer design.
Cons:
Primer design requires precision for correct overlap and orientation.
Efficiency can depend on factors like fragment size and sequence complexity.
Commercial kits can be expensive.
Tips and Tricks:
Optimal Overlap Length: Overlaps of 15–25 nucleotides typically provide efficient annealing, though larger fragments may require longer overlaps.
High Purity DNA: DNA fragments should be pure, often achieved by gel purification post-PCR, for optimal results.
Control Reactions: Always include controls (like no-insert controls) to detect potential background noise or unwanted ligation.
Q: What plasmid quality control (QC) tests are performed?
A: For research-grade plasmids, we perform the QC tests listed below.
- Appearance: A visual inspection of the plasmid solution to assess its clarity and the absence of particles or discoloration.
- A260/280: Measures the purity of plasmid DNA by comparing absorbance at 260 nm (nucleic acids) and 280 nm (proteins). A ratio of ~1.8 indicates pure DNA.
- Homogeneity by Agarose Gel: Assesses the uniformity of plasmid DNA by running it on an agarose gel to ensure consistent molecular weight and the ratio of supercoiled plasmids.
- Restriction Analysis: Verifies the identity and integrity of plasmid DNA by cutting it with specific restriction enzymes and analyzing the resulting fragments via gel electrophoresis.
- Endotoxin by LAL: Detects bacterial endotoxins in plasmid preparations using the Limulus Amebocyte Lysate (LAL) assay, ensuring plasmids are safe for sensitive applications.
Additionally, upon request, we offer extra QC tests, which are also included in our preclinical plasmid quality control.
- Homogeneity by HPLC: Uses High-Performance Liquid Chromatography (HPLC) to evaluate the uniformity and purity of plasmid DNA, and measure the ratio of supercoiled plasmid DNA.
- Residual RNA by SYBRGold: Quantifies any remaining RNA in the plasmid preparation by staining with SYBRGold and analyzing fluorescence intensity.
- Residual E. coli DNA by qPCR: Detects and quantifies residual E. coli genomic DNA in plasmid preparations using quantitative PCR (qPCR).
- Bioburden Testing by Direct Inoculation: Assesses the microbial contamination level of the plasmid preparation by inoculating samples in growth media and monitoring for microbial growth.
- Sequencing by Sanger: Confirms the accuracy of the plasmid sequence by using Sanger sequencing to verify the inserted DNA or the entire plasmid.
- Residual Host Protein by ELISA: Detects any leftover host proteins in plasmid preparations using an ELISA assay specific to E. coli proteins.
- Mycoplasma Contamination by qPCR: Screens for mycoplasma contamination in the plasmid sample using sensitive qPCR techniques.
- pH by Potentiometry: Measures the pH of the plasmid solution to ensure it is within the acceptable range for stability and application.
- Residual Kanamycin by ELISA: Detects any remaining kanamycin antibiotic from the plasmid selection process using an ELISA assay.
- Sterility: Confirms the absence of viable microorganisms in the plasmid preparation, ensuring it is sterile and suitable for sensitive applications.
- Osmolality: Measures the osmolality (concentration of solutes) in the plasmid solution to ensure it is within acceptable limits for biological compatibility.
Q: Are pH measurements required, and is a large amount of sample wasted to carry out pH measurements?
A: Measurement of pH is a mandatory for the release of rAAV Fast Service deliverables. A micro pH electrode may be used to save sample and thus the required sample volume to perform pH measurements is only ~15uL-100uL.
Q: What is loading?
A: In accordance with the Pharmacopoeia General Rules 0942, we use the minimum filling quantity inspection method for detecting sample loading quantity.
Q: How to interpret A260/A280 value?
A: A260/A280 is the ratio of sample absorbance measured at wavelengths of 260nm and 280nm. This measure is commonly thought to represent the ratio of DNA to protein in a sample. For rAAV, A260/A280 can used as a measure of the full to empty shell rate and to identify protein contamination. Low A260/A280 levels may suggest that the empty shell rate is high. Alternatively, high A260/A280 may suggest that the sample has been contaminated with proteins that are not incorporated into the AAV capsid shell. The greatest advantages of this measure are its convenience and speed.
Q: What tests are performed to differentiate rAAV capsid proteins from specific protein impurities?
A: SDS-PAGE is used to identify rAAV capsid proteins. In addition, SDS-PAGE can be used to directly identify specific protein impurities including the presence of host proteins, BSA, or degraded AAV capsid proteins.
Q: How is rAAV infection titer measured, given that rAAV does not integrate the host genome?
A: The current standard for determining infection titer is TCID50 (Median Tissue Culture Infectious Dose). In this assay we test the infection rates of rAAV sample serial dilutions in H5 cells. This initial test is then followed by qPCR detection of the rAAV genome.
Q: If I have determined that a rAAV DNA vector reliably drives transgene expression in host cells prior to rAAV packaging, can I assume that this DNA vector will also drive transgene expression after it is packaged into an rAAV that is then used to infect cells?
A: Successful transgene expression via rAAV infection relies on several factors beyond the functionality of the DNA vector. We therefore recommend that independent validation of transgene expression is performed for all packaged rAAV.
Q: What does rcAAV refer to, and are there regulations regarding rcAAV content in GMP rAAV samples?
A: The term rcAAV stands for replication-competent AAV. In most cases rAAVs are designed to be replication incompetent. Thus, rAAV samples should not contain rcAAV and regulations require negligible replication capability for GMP rAAV samples.
Q: What is the difference between visible foreign matter and insoluble particles?
A: Visible foreign matter can be identified by visual inspection. According to pharmacopoeia general rule 0904, visible foreign matter in injections, ophthalmic liquid preparations, and sterile APIs can be visually observed under specified conditions . Visible foreign matter is usually derived from insoluble particles of a size or length is greater than 50μm.
Insoluble particles between 10μm and 50μm cannot be seen with the unaided eye and must therefore be detected by instrumentation. Insoluble particles identification is commonly carried out with an insoluble particle analyzer. According to pharmacopoeia general rule 0903, evaluation of the size and quantity of insoluble particles within materials to be delivered by intravenous injection (solution injection, sterile powder for injection, concentrated solution for injection) can be made according to the project nature.
Q: If I have successfully carried out several in vivo experiments in mice may I assume that mycoplasma and bacterial endotoxin levels are within acceptable ranges and forgo direct testing for these contaminants?
A: Reagents used for Gene Therapies should abide by all domestic regulations in the “Guiding Principles for Quality Control of Human Recombinant DNA Products” by the EDC. Under these standards Mycoplasma and endotoxin testing is required.
Q: What is the source of PackGene cell bank?
A: PackGene’s h293 cell bank is officially authorized for commercial use.
Q: What is the AAV output for a single batch fermentation?
A: PackGene offers single batch fermentation at several volumes, including: 2L, 7L, 25L, 50L, and 200L. AAV yields for each of these production volumes varies across AAV serotypes. As an example, AAV9 is a medium to high-yielding serotype, and expected yields for AAV9 are as follows:
| Expected yield for AAV9 | |
| Volume | Yield |
| 2L | 1E+14GC |
| 50L | 1E+16GC |
| 100L | 2E+16GC |
Q: How are GC/ml and vg/ml related to one another, and how does PackGene determine GC/ml for AAV products?
A: The terms genome copies per ml (GC/ml) and viral genomes per ml (vg/ml) are interchangeable and equal in most cases. At PackGene we may test GC by both qPCR and ddPCR. Testing by qPCR involves the use of a calibration standard while ddPCR may use optional reference products. Measurements by qPCR are more likely to be influenced by inter-lab and inter-operator variables, and ddPCR generally shows lower %RSD precision. Typically, GC is determined by qPCR during process exploration phase and for intermediate products while the GC of final products is more often determined by ddPCR.
Q: Do specific primers need to be designed to determine GC/ml for a custom AAVs?
A: Genome copies per ml (GC/ml) can be determined using primers directed at common vector elements during the early development stage, and thus we use common features such polyA segments or ITRs to determine GC/ml during this stage. However, it is recommended that specific primers targeted at AAV vector transgenes are used for testing the GC of GMP products. Our final fast service titer test is designed to use transgenes specific primers.
Q: What method is used to determine the empty shell rate for AAV samples?
A: Viral empty shell rate can be determined using several techniques including anion chromatography HPLC, Analytical Ultracentrifugation (AUC), Transmission Electron Microscopy (TEM), CyroTEM, or VG Titer/Capsid titer. AUC, TEM and CyroTEM are typically not suitable for quantitative quality control determinations and thus PackGene’s standard method for empty shell rate determination is anion chromatography HPLC. PackGene can provide additional CyroTEM and AUC analytical services to serve as a secondary verification of the results derived from anion chromatography HPLC.
Q: What is the expected empty shell rate PackGene AAV Fast Services?
A: The expected empty shell rate for our AAV Fast Services varies across AAV serotypes. As one example, the expected empty shell rate for AAV9 generated through our AAV Fast Service is lower than 10%.
Q: Is verification of cell banks in accordance with the Chinese Pharmacopoeia 2015 Three General Principles “The Preparation and Verification Regulations of Animal Cell Matrix for the Verification of Biological Products”?
A: Yes.
Q: HIV, HBV, HCV testing, pyrogen-free tests, and wild virus tests are not in the standard quality control services list. Do these tests need to be applied?
A: The harvest liquid generated through PackGene’s AAV fast service will be broadly tested for exogenous viral elements as necessary following an evaluation of project characteristics. Both the original harvest liquid and final deliverables for the AAV Fast service will be devoid of such exogenous viral elements.
Q: What does PackGene consider acceptable error or deviation ranges for standard QC analysis methods?
A: The %RSD of general biochemical methods is usually 15%-25%, and the accepted standard of ddPCR is 10%. In most cases the %RSD for PackGene’s QC ddPCR verification data is lower than 5%, and the %RSD of TCID50 is lower than 10%.
Q: Are there any requirements for the number of samples sent in batches?
A: It is recommended that the sample volume of a single commissioned express delivery is not less than 50uL to avoid the effects of freezing and thawing, evaporation, and tube wall adhesion. The recommended sample delivery volume for genome titer, plasmid DNA residue, rcAAV, etc. is more than 10ul. The recommended sample delivery volume for infection titer is more than 20ul, and the sample delivery amount for empty shell rate testing is 5E+13vg.