One of the key factors underlying the design of any successful experiment is the choice of the vector system used for delivering the genes of interest into the target cells. Given that there are various viral and non-viral vector options available, several factors should be taken into consideration while selecting the ideal vector suitable for your experimental design. Some of the key considerations include: Are your target cells easy or difficult to transfect? Do you want transient expression or stable integration into the host genome? Do you need to use a customized promoter to drive your gene of interest? Will your vector be used in cell culture or in vivo? Do you need conditional or inducible gene expression? How big is your gene of interest?'
The table below lists the commonly used vector systems and key considerations for selecting the right vector suitable for your experimental design.
|Regular plasmid vectors||Viral vectors||Transposon-based vectors|
|Transient expression or stable integration||Transient||Stable integration||Stable integration|
|Cargo capacity||Large||Small to medium||Medium to large|
|Primary use||Cell culture||Cell culture & in vivo||Cell culture & in vivo|
|Promoter customization||Yes||Depending on viral vector type||Yes|
Technical simplicity: Regular plasmid vectors rely on simple transfection-based methods for the delivery of target genes into host cells. Delivering plasmid vectors into cells by conventional transfection is technically straightforward, and far easier than virus-based vectors which require the packaging of live virus.
Large cargo capacity: Our regular plasmid vectors have a large cargo capacity of ~30 kb. This provides plenty of room to add variable vector components such as the user’s gene of interest, a promoter and a marker unlike viral vectors majority of which have a moderate to limited cargo capacity.Disadvantages
Non-integration of vector DNA: Conventional transfection of plasmid vectors is also referred to as transient transfection because the vector stays mostly as episomal DNA in cells without integration. However, plasmid DNA can integrate permanently into the host genome at a very low frequency (one per 102 to 106 cells depending on cell type). If a drug resistance or fluorescence marker is incorporated into the plasmid, cells stably integrating the plasmid can be derived by drug selection or cell sorting after extended culture.
Limited cell type range: The efficiency of plasmid transfection can vary greatly from cell type to cell type. Non-dividing cells are often more difficult to transfect than dividing cells, and primary cells are often harder to transfect than immortalized cell lines. Some important cell types, such as neurons and pancreatic β cells, are notoriously difficult to transfect. Additionally, plasmid transfection is largely limited to in vitro applications and rarely used in vivo.
Non-uniformity of gene delivery: Although a successful transfection can result in very high average copy number of the transfected plasmid vector per cell, this can be highly non-uniform. Some cells can carry many copies while others carry very few or none. This is unlike transduction by virus-based vectors which tends to result in relatively uniform gene delivery into cells.
Suitable for difficult-to-transfect cells: Viral vectors are the preferred method of gene delivery for difficult-to-transfect cell lines. Most viral vectors can transduce a wide variety of mammalian cell lines due to the broad tropism conferred by the viral envelop proteins. Our lentivirus packaging system adds the VSV-G envelop protein to the viral surface which has a very broad tropism. As a result, cells from all commonly used mammalian species (and even some non-mammalian species) can be transduced. Furthermore, almost any mammalian cell type can be transduced (e.g. dividing cells and non-dividing cells, primary cells and established cell lines, stem cells and differentiated cells, adherent cells and non-adherent cells). Neurons, which are often impervious to conventional transfection, can be readily transduced by our lentiviral vector.
Similarly, when our AAV vectors are packaged into virus, different serotypes can be conferred to the virus by using different capsid proteins for the packaging. Different serotypes can render the virus with different tissue tropism (i.e. tissue specificity of infection). A wide range of cell and tissue types from commonly used mammalian species such as human, mouse and rat can be readily transduced with our AAV vector when it is packaged into the appropriate serotype.
Suitable for in vitro and in vivo applications: Viral vectors can be used for effective transduction of cultured cells as well as live animals unlike regular plasmids which are commonly used for in vitro applications.
Relative uniformity of gene delivery: Generally, viral transduction can deliver vectors into cells in a relatively uniform manner. In contrast, conventional transfection of plasmid vectors can be highly non-uniform, with some cells receiving a lot of copies while other cells receiving few copies or none.Disadvantages
Medium to small cargo capacity: Most viral vectors have a limited cargo capacity when compared to regular plasmid or transposon-based vectors. It is important to take the cargo capacity into consideration while designing a viral vector since exceeding the viral vector capacity often adversely affects the virus packaging process. The table below shows the upper limit of viral genome for different viral vectors.
|Virus Type||Upper Limit of Viral Genome||Upper Limit of User’s DNA Fragment|
|Lentivirus||9.2 kb (from 5’ LTR-ΔU3 to 3’ LTR-ΔU3)||6.4 kb|
|Adenovirus||38.7 kb (from 5’ ITR to 3’ ITR)||7.5 kb|
|Adeno-associated virus||4.7 kb (from 5’ ITR to 3’ ITR)||4.2 kb|
Technical complexity: The use of viral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technically demanding and time consuming relative to conventional plasmid transfection.Viral vector types
Common viral vectors used in biomedical research include lentivirus, adeno-associated virus (AAV), and adenovirus each with its advantages and disadvantages. The table below lists key factors that should be taken into consideration while selecting the right viral vector for your experiment.
|Tropism||Broad||Depending on viral serotype||Ineffective for some cells|
|Can infect non-dividing cells?||Yes||Yes||Yes|
|Stable integration or transient?||Stable integration||Transient, episomal||Transient, episomal|
|Maximum titer||High||High||Very high|
|Primary use||Cell culture and in vivo||In vivo||In vivo|
|Immune response in vivo||Low||Very low||High|
Permanent integration of vector DNA: Transposon-based vectors rely on conventional transfection for the delivery of target genes into host cells. Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, transfection of mammalian cells with transposon-based vectors along with the corresponding helper plasmid can deliver genes carried on the transposon permanently into host cells due to the integration of the transposon into the host genome.
Technical simplicity: Delivering plasmid vectors into cells by conventional transfection is technically straightforward, and far easier than virus-based vectors which require the packaging of live virus.Disadvantages
Limited cell type range: The delivery of transposon-based vectors into cells relies on transfection. The efficiency of transfection can vary greatly from cell type to cell type. Non-dividing cells are often more difficult to transfect than dividing cells, and primary cells are often harder to transfect than immortalized cell lines. Some important cell types, such as neurons and pancreatic β cells, are notoriously difficult to transfect. Additionally, plasmid transfection is largely limited to in vitro applications and rarely used in vivo. These issues limit the use of transposon-based vector systems.
Choosing an appropriate selection marker is a critical step while designing a vector for ensuring experimental success. A selection marker enables the experimenter to successfully identify cells that have been positively transfected or transduced with the vector.
Two types of markers are commonly used for selection of positively transfected/transduced cells – drug-selection markers and fluorescent protein markers. VectorBuilder offers a variety of drug-selection markers on its vectors, such as puromycin (Puro), neomycin (Neo), hygromycin B (Hygro) and blasticidin (Bsd) which function by conferring resistance against the respective antibiotic in cells carrying the vector with the marker. Cells not carrying the vector on the other hand are non-resistant to the antibiotic and are therefore, killed in its presence. Fluorescent markers on the other hand enable researchers to select for positively transfected/transduced cells by visualization under a fluorescence microscope or by fluorescence activated cell sorting (FACs).
Sensitivity towards a drug-selection marker can vary significantly from one cell type to another. While some cell types may naturally have some degree of resistance to certain antibiotics without the resistance gene, certain cell types are sensitive to certain antibiotics even with the resistance gene. Therefore, different drug-selection markers should be tested to find the optimal one for your cell type. In general, we have found that puromycin kills non-resistant cells faster and more consistently than other antibiotics. For this reason, we recommend puromycin for most cell types.
Once the optimal antibiotic has been selected, it is also important to determine the optimal antibiotic concentration and the duration of selection required to kill the target cells by performing an antibiotic kill curve. This ensures the presence of all positively transfected/transduced cells in the experimental cell population, thereby reducing variability and helps in the establishment of a stable cell line if needed for experiments requiring long-term gene expression. The table below lists the recommended concentration and selection duration of commonly used drugs in common cell lines.
|Antibiotics||Cell line||Recommended concentration||Recommended duration|
|Puromycin||293T||1-2 ug/ml||3-5 days|
|Geneticin (G418)||HT1080||500-1000 ug/ml||7-11 days|
|Blasticidin||293T||5-15 ug/ml||7-11 days|
|Hygromycin B||293T||100-200 ug/ml||5-7 days|
While drug-selection markers are suitable for experiments requiring long-term gene expression through the establishment of a stable cell population which can be time consuming, fluorescent markers on the other hand provide a relatively simple and quicker alternative for selecting positively transfected/transduced cells by visualization under a fluorescence microscope. Selection of an ideal fluorescent marker for your vector depends on several factors such as whether it is single-color or multi-color experiment, brightness of the fluorescent protein, maturation time of the protein and sensitivity of the target cells towards toxicity and protein aggregation observed with certain fluorescent proteins.
While incorporating either a drug-selection or a fluorescent marker in the vector is the most commonly used approach, adding a dual-selection cassette consisting of both a drug-selection marker as well as a fluorescent marker provides a highly versatile and efficient method for selecting positively transfected or transduced cells. However, the use of a dual-selection cassette is sometimes restricted by the limited cargo capacity of the vector being used such as observed with viral vectors. VectorBuilder offers a variety of single as well as dual-selection cassettes to provide you with the flexibility to choose the right selection marker suitable for your experiment.
When using shRNAs, it is important to recognize the fact that not all shRNAs will work. Knockdown effects of empirically designed shRNAs are often limited by variations in specificity and efficiency observed from one shRNA to another. The potency of an shRNA is determined by several factors including length of the shRNA, loop structure, GC profile and thermodynamic stability of the shRNA, secondary structure of the target sequence and off-target matches to other genes. Typically, ~50-70% of shRNAs have noticeable knockdown effect, and ~20-30% of them have strong knockdown. Therefore, it is important to test multiple shRNAs to find the most potent shRNA for knocking down your gene of interest.
Based on our experience and feedback from our customers, we know that generally when 3 or 4 shRNAs are tested for any arbitrary gene, typically 2 or 3 produce reasonable to good knockdown. If you try a few shRNAs targeting a specific gene, it is possible that by chance, none will produce satisfactory knockdown. When this happens, the best approach is to try more shRNAs, especially the ones that have literature validation. Many researchers also use a “cocktail” of shRNAs (i.e. mixture of different shRNAs) targeting the same gene, which sometimes can improve knockdown efficiency.
VectorBuilder has created shRNA databases that contain optimized shRNAs for common species. shRNAs in these databases are designed and scored applying rules like those used by the RNAi consortium. When you design shRNA vectors using our online design tool, you will have the option to search your target genes in our database. Once you enter your genes of interest, you will see detailed information of all available shRNAs we have designed for you, including a link to UCSC Genome Browser to view these shRNAs in the context of their genomic sequence and all the transcript isoforms. Our database ranks all available shRNAs for a target gene in order of their decreasing knockdown scores and recommends testing the top 3 shRNAs with the highest knockdown scores.
Lentiviral vectors are the most preferred shRNA delivery vehicle for applications requiring long-term knockdown of gene expression. However, several researchers prefer to use regular plasmid vectors for transient transfection and validation of candidate shRNAs targeting their genes of interest to minimize higher costs and technical complexities associated with lentiviral vector-based methods. Once the shRNA(s) with the highest knockdown efficiency is identified, it can then be expressed using a lentiviral vector for achieving stable knockdown of the target gene.
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