Gene Overexpression
PackGene’s AAV gene overexpression plasmid allows you to customize various elements, including:
- Promoters (universal or tissue-specific)
- Gene of Interest (GOI)
- 3’ Regulatory Elements
- Poly A Signals
Additionally, you can incorporate a secondary reporter expression cassette with fluorescent proteins, provided the total gene length stays within the AAV genome size limit. Reporters such as EGFP and mCherry, along with protein tags like HA, Flag, and His, can be easily selected and inserted into the plasmid framework using our piVector Designer. Our extensive library of promoters and GOIs further enhances the customization options available.
CRISPR Gene editing
PackGene’s proprietary AAV Cas9 expression vectors have been carefully designed to co-express SpCas9 or SaCas9, sgRNA, or both Cas9 and sgRNA from a single vector.
Available in piVector Designer
- SpCas9: dual vector or all-in-one vector
PackGene’s proprietary SpCas9 expression vectors are engineered to co-express SpCas9 and sgRNA within a single AAV vector. To ensure that the SpCas9 expression sequence stays within the AAV vector insert length limit of ~5Kb, we use the compact and efficient promoter sequences EF1s or miniCMV. For sgRNA expression, we use the efficient and relatively short H1 promoter to reduce the total bp length of the sgRNA expression cassette. With this strategy, we have generated a single AAV CRISPR vector capable of reliable and efficient SpCas9 and sgRNA co-expression, minimizing the risk of mixed cell expression phenotypes and the potential for inconsistent or spurious data. - SaCas9: dual vector or all-in-one vector
SaCas9 is a shorter version of Cas9 that maintains similar cleavage activity to SpCas9 but is encoded by a shorter gene of approximately 3.2 kb compared to SpCas9’s ~4.2 kb. In addition, the SaCas9 PAM sequence (NNGRRT) occurs less frequently in host genomes, approximately once every 32 bp, and the 21-23 nt target sequence is longer than that of traditional SpCas9. This reduced PAM frequency and extended target sequence length contribute to SaCas9’s higher binding specificity, theoretically resulting in a lower off-target cleavage rate compared to traditional SpCas9.
Available upon request, please contact our technical support team
- SpCas9HF
Hi-fidelity version of SpCas9 - SaCas9HF
Hi-fidelity version of SaCas9 - AAV-CRISPR gene activation MS2-P65-HSF1
While Cas9 is typically paired with traditional sgRNA to produce targeted gene knockouts, it can also be paired with a sgRNA containing a modified MS2 RNA linker scaffold for an alternative function. In the modified MS2 configuration, SpCas9 loses its DNA cleavage capability and instead recruits the MS2-P65-HSF1 transcriptional activation complex, which drives downstream gene transcription. This approach enables transcriptional activation, potentially increasing endogenous gene expression by more than 1000-fold. - NmCas9
The 3.3-kb-long NmCas9 is an alternative version of Cas9 with similar activity to SpCas9 but offers an advantage in applications where exceptionally low off-target cleavage rates are required. NmCas9 recognizes a longer PAM sequence (NNNNGATT), which occurs less frequently than the SpCas9 PAM at approximately once every 128 bp. Combined with its extended target sequence length of 21-23 nt, this results in higher target specificity. These characteristics contribute to NmCas9’s theoretically lower off-target cleavage rate compared to other Cas9 variants, making it ideal for highly precise genome editing applications. - AsCpf1 and LbCpf1
While Cas9 and its variants are the most commonly used CRISPR effector endonucleases, the Cpf1 endonuclease has gained increasing popularity as an alternative for CRISPR gene editing. Similar to Cas9, Cpf1 requires a gRNA for activation, binds genomic DNA adjacent to a PAM sequence, and cleaves DNA following sgRNA target sequence annealing. However, Cpf1 is shorter in size than Cas9, and requires a shorter sgRNA for full functionality. Another key distinction is that while Cas9 generates blunt-ended DNA cuts, Cpf1 produces 4-5 base pair protruding sticky ends, facilitating low-error and controllable gene insertions.
What is CRISPR?
CRISPR is a state-of-the-art genetic modification tool that can be used to knockout, mutate, or overexpress a gene of interest. CRISPR-based research strategies have been widely employed across biological fields of study due to their powerful performance and relatively straightforward mechanism of action.
CRISPR gene editing requires three components: (1) a guide RNA (gRNA), (2) a Cas9 endonuclease, and (3) a protospacer adjacent motif (PAM) within the target genome. The gRNA is a single stranded RNA molecule consisting of two regions: (1) a gene-targeting sequence that complements a 15-24 base pair segment of the gene of interest, and (2) a scaffold region that binds to Cas9. The gRNA scaffold region binds to a Cas9 endonuclease while leaving the host DNA compliment region of the gRNA unbound. The Cas9-gRNA complex may then scan the host cell’s genomic DNA for a specific 2-6 base pair sequence referred to as the PAM. Once the Cas9-gRNA complex recognizes the PAM sequence the gene targeting region of the gRNA may then anneal to the host DNA adjacent to the PAM sequence. After successful binding of the gRNA gene targeting region Cas9 will cleave the host DNA adjacent to the PAM sequence.
Cas9 mediated DNA cleavage generally results in gross disruptions in gene transcription and functional gene knockout following DNA repair. Alternatively, cleavage can be used to introduce precise DNA mutations or to activate gene expression using a modified gRNA scaffold region that inhibits Cas9 cleavage capabilities while simultaneously recruiting transcriptional proteins to a gene’s promoter region. Importantly, the specificity of target DNA recognition makes it feasible to design CRISPR based gene editing strategies targeted at nearly any gene.
The design and production of AAV vectors for CRISPR based gene editing can be both challenging and time-consuming. However, our professional team has experience and knowledge necessary to deliver high quality CRISPR-AAVs that are ready to use for your experimental needs immediately on delivery.
shRNA
Available in piVector Designer
Pol III Promoter driven shRNA | miR30-Based shRNA
RNA interference (RNAi) is a highly specific tool for protein knockdown, where small non-coding RNAs bind to a target proteins mRNA, promoting its degradation and consiquently reducing protein translation. RNAi mechanisms are endogenously present in most eukaryotes, and have been widely adopted as a molecular tool for gene function studies, drug discovery, and gene silencing therapy.
Recent clinical trials have demonstrated that RNAi hold significant potential in targeting disease-causing genes for the treatment of human diseases. In comparison to other delivery methods, AAV vector-based expression of RNAi has been shown to drive prolonged reduction of mRNA targets in live animals.
miRNA
PackGene offers custom inhibitory miRNA AAV vectors designed to inhibit endogenous miRNA expression and function in live animals. AAV-miRNA cloning ensures greater expression durability and longer effect duration compared to traditional synthetic miRNA inhibitors while also improving safety and minimizing immunogenicity.
miRNA are small non-coding RNAs that are endogenously expressed in eukaryotes and play important role in regulating gene expression. miRNA inhibitors are RNA molecules that are designed as reverse compliment sequence to endogenous miRNA and functionally block their gene regulation capabilities.
Our service offers direct, convenient, and efficient expression of miRNA inhibitors for in vivo animal experiments, allowing for targeted delivery to specific organs and tissues via custom AAV serotypes. This approach ensures greater expression durability and longer effect duration compared to traditional synthetic miRNA inhibitors, while also increasing safety and reducing immunogenicity.
What are the components of plasmid vectors?
Plasmid vectors are indispensable in genetics and molecular biology, serving as vehicles to insert, manipulate, and transfer genes within organisms. They typically contain key components like the origin of replication (ori), promoter regions, open reading frames (ORFs), regulatory elements, a polyadenylation (poly A) signal, multiple cloning sites (MCS), and antibiotic resistance genes. Choosing the right combination of these elements is crucial for the success of your research, as each one plays a specific role in plasmid functionality.
What bacteria strains do you use for cloning and plasmid preparation?
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.
How is a plasmid constructed based on your design?
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.
What plasmid quality control (QC) tests are performed?
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 Kan by ELISA: Detects any remaining kan 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.