FIV Lentivectors

What is a “lentiviral” vector and how is this different from a standard cloning vector?

A lentiviral vector contains the genetic elements that enable a copy of it to be packaged into a viral particle when it is present in a cell line that expresses the other viral particle components, such as the coat proteins (i.e., a “packaging” cell line). For example, a lentiviral vector contains the critical portions of the “long terminal repeat” (LTR) sequences, which are necessary to express the packaged portion of the vector. Standard vectors cannot be packaged into viral particles.

The advantage of packaging the vector into a viral particle is transduction (infection with biologically-inactive viral particles) is a much more effective way to deliver an expression construct, than typical transfection methods. When SBI’s pFIV lentiviral expression vectors are packaged into VSV-G pseudotyped viral particles (using the pFIV-PACK System) they robustly transduce almost any mammalian cells, including non-dividing cells and whole model organisms. This capability makes this system much more effective than standard expression vectors for delivering expression cassettes.

What is the difference between SBI’s pFIV lentiviral-based cloning and expression vectors and other viral-based vectors?

The difference between lentiviral-based vectors and adenoviral vectors
Like lentiviral-based vectors, adenoviral vectors can be packaged into viral particles when expressed cells expressing the appropriate genetic elements, and like lentiviral particles, adenoviral particles will infect a wide range of cells, including non-dividing and dividing cells. However, once introduced into target cells, adenoviral expression is very high and often has toxic effects on the cell. Obviously, this type of system is not suitable for gene function analysis since it would not be clear whether observed effects are a result of the expressed sequence or side effects of over expression.

The difference between lentiviral-based vectors and other retroviral-based vectors
SBI’s pFIV lentiviral system is based on the feline immunodeficiency (FIV) virus and the pFIV vectors, when packaged into VSV-G pseudotyped viral particles, effectively transduce both dividing and non-dividing cells. Most other commercial retroviral vectors, for example vectors from system based on the murine leukemia virus, are only effective and transducing actively dividing cells. This limitation severely limits the utility of these other retroviral-based vectors for expression of siRNA for functional analysis.

The difference between pFIV lentiviral-based vectors and HIV-based vectors
The pFIV vectors are based on the feline immunodeficiency (FIV) retrovirus. Despite common close exposure of humans to FIV through contact with domestic cats (including bites, scratches, etc.), no human infection or disease has ever been associated with FIV. Of course, SBI’s pFIV Vectors are also self-inactivating as a result of a deletion in the U3 region of 3’-ΔLTR. Upon integration into the genome, the 5’ ΔLTR promoter is inactivated, which prevents formation of replication-competent viral particles. Although commercially available HIV-based vectors are similarly self-inactivating, HIV cloning vectors can pose a slight biohazard risk due to possible recombination with endogenous viral sequences to form a self-replicating HIV virus.

What is the difference between SBI’s pFIV Lentiviral siRNA Cloning and Expression Vectors and other siRNA cloning and expression vectors on the market?

How are they different from non-viral based siRNA vectors?
There are a number of non-viral-based siRNA cloning systems on the market (Clontech, Ambion, etc.). As explained in the previous section, lentiviral-based vectors can be packaged into a viral particle and robustly and efficiently introduced into any type of mammalian cells, including non-dividing cells and difficult-to-transfect cells, such as neuronal, blood, primary, and stem cells.

How are they different from other viral-based siRNA vectors on the market?
Currently, we are only aware of Invitrogen’s HIV-based RNAi Expression System. As opposed to our FIV-based expression system, Invitrogen’s system is complicated by extra steps, requires the end user to clone their siRNA insert into the pENTR/U6, and then perform a recombination reaction between the pENTR/U6 entry construct and the pLenti6/BLOCK-iT-DEST to generate the pLenti6/BLOCK-iT expression construct. In addition to the extra steps and time required, Invitrogen’s Lentiviral vectors and packaging system are 3 times more costly. This does not include the increased costs of using the Gateway reagents (as compared to the costs of standard cloning reagents used by SBI’s Expression System) required through their use of the Gateway cloning system.

The unique pFIV Double-Promoter siRNA vector.

SBI is the only company to offer a double promoter vector that expresses siRNA directly. Instead of cloning a much longer template sequence that expresses a hairpin siRNA, the double promoter vectors only require the cloning of a short template siRNA sequence. The double promoter also provides higher stability of siRNA template inserts during propagation in E. coli since it does not contain a hairpin structure that is often removed during bacterial replication. This increased stability is very critical for construction of representative high complexity siRNA libraries. In addition, shorter siRNA sequences are less costly to produce and are easier to clone. These advantages make the double promoter siRNA Vectors, like the pFIV-H1/U6-Puro, highly suitable for siRNA library construction, as well as convenient for cloning and expressing single siRNA sequences.

What is copGFP?
The copGFP protein is a novel natural green monomeric GFP-like protein from copepod (Pontellina sp.). copGFP is a non-toxic, non-aggregating protein with fast protein maturation, high stability at a wide range of pH (pH 4-12) and does not require any additional cofactors or substrates.

What is the difference between copGFP and EGFP?
Due to its exceptional properties, copGFP is an excellent fluorescent marker that can be used instead of EGFP (the widely used Aequrea victoria GFP mutant) for monitoring delivery of FIV constructs into cells. The copGFP protein has a very bright fluorescence that exceeds, at least, 1.3 times the brightness of EGFP.

What are the copGFP fluorescent characteristics?
copGFP emits green fluorescence with the following characteristics:

emmision wavelength max – 502 nm;
excitation wavelength max – 482 nm;
quantum yield – 0.6;
extinction coefficient – 70,000 M-1cm-1.

Can I create cells stably expressing a particular siRNA with the pFIV-copGFP Vectors?
Yes, the pFIV-copGFP Vectors contain all the genetic elements responsible for packaging, transduction, stable integration of the viral expression construct into genomic DNA, and expression of the siRNA effector sequence. The pFIV-PACK Lentiviral Vector Packaging Kit (LV100A-1) provides all the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant viral particles.

When the construct is expressed in the packaging cell line (HEK 293T cells that stably express the SV40 large T antigen), the pFIV transcripts are efficiently packaged into pseudoviral particles. Following transduction into target cells, the expression cassette is reverse transcribed and integrated into the genome of the target cell. Cells stably expressing the siRNA construct can be selected by FACS.

What are the safety guidelines for working with the pFIV Vectors?
Use of FIV-based vectors falls within the NIH Biosafety Level 2 criteria. A detailed description of laboratory Biosafety level criteria can be found on the Centers of Disease Control Office of Health and Safety web site:

http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4s3.htm
http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm

The safety guidelines for working with the pFIV vectors can also be found in section I of the user manual.

Is there a possibility of generating self-replicating viral sequences?
These vectors have been modified to remove sequences that overlap with the packaging plasmid to minimize the possibility for homologous recombination and generation of self-replicating viral sequences when co-transfecting these constructs into packaging cells. The pFIV-H1-Puro and pFIV-H1/U6-Puro vectors also have a deletion in the enhancer of the U3 region of 3’-LTR to ensure self-inactivation of the lentiviral vector after transduction and integration of the sequences into the genomic DNA of the target cells.

Do the pFIV Vectors work for in vivo experiments?
pFIV Vectors can be used for in vivo experiments by generating transgenic gene-specific knockdowns that target any gene of your choice. The pFIV-PACK Packaging Vector provides all the necessary coat proteins to manufacture pseudoviral particles for delivery of the siRNA expression construct into any mammalian cells or model organisms, such as transgenic mice.

What does the Lentiviral expression vector contain?

The Lentiviral expression vectors contain all the genetic elements responsible for packaging, transduction, stable integration of the viral expression construct into genomic DNA, and expression of the siRNA effector sequence.

What does the Lentiviral packaging vector contain (pFIV-PACK)? Do I need the pFIV-PACK packaging vector?
The packaging vector provides all the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant viral particles. To produce a high titer of viral particles, transiently co-transfect packaging cells (HEK 293T cells) with the expression and packaging vectors.


What is the difference between the pFIV-H1 Vector and the pFIV-H1/U6 Vector?
SBI offers two vectors for the cloning and expression of siRNA. The pFIV-H1 vector is an approach that utilizes a single siRNA sequence with a fold-back stem-loop structure (also known as a “hairpin”) that is expressed from a single promoter (H1). The sequence is then converted to a double-stranded siRNA after intracellular processing cleaves the loop.

The pFIV-H1/U6-Puro vector is an approach in which the sense and anti-sense strands are transcribed separately from two independent promoters (H1 and U6) and form the siRNA duplex.

A more detailed description of each system is available at www.systembio.com

What does FIV stand for?
Feline Immunodeficiency Virus

Is there a limit in size for the siRNA template?
Although there is no standard rule, SBI recommends a 19-29 nucleotide in length sequence for the siRNA template. 25-27 nucleotide siRNAs have a slightly better silencing efficiency than shorter oligos, but 19 nucleotide oligos are more commonly used.

 

Full Spectrum Complete Transcriptome RNA Amplification Kit

How are your Universal Primers different from random primers?
As PCR primers, random primers are very inefficient and will prime off the ribosomal RNA. This leads to cDNA that is comprised mostly of “junk” sequences. SBI’s Universal Primer is designed for true mRNA priming and is very efficient.

Are partial cDNAs generated?
The cDNA generated by the Universal Primer will generate partial cDNAs throughout the entire length of the transcripts. However, the cDNAs generated will be overlapping cDNA fragments encompassing the entire gene.

What is the required starting amount of RNA?
We recommend starting with approximately 100 ng of total RNA, and a minimum starting amount of 5 ng of total RNA.

Does the Full Spectrum Global Amplification Kit work on Prokaryotic systems?
SBI’s amplification system utilizes a mixture of non-redundant primers that have been selected to amplify mRNA. The system should be very efficient on non-poly A mRNAs, such as bacterial mRNAs.

Does your system work with partially degraded RNA?
Experiments have shown SBI’s system works with partially degraded RNA.

Does your system work with paraffin embedded tissue?
Applications regarding use of RNA from paraffin embedded tissue are currently in development.

What yield should I expect?
The Full Spectrum Global Amplification Kit will yield 2-5 ug of stable cDNA product ready for analysis using quantitative PCR. As a note, if using a starting amount of 5ng, expect a yield of 1-2 ug.

Is there a 3’ bias?
The Full Spectrum RNA Amplification utilizes a mix of non-degenerate primers that are designed to randomly amplify and will generate partial cDNAs throughout the entire length of the transcripts. The cDNAs generated will be overlapping cDNA fragments encompassing the entire gene and there will be no bias toward the 3' or 5' ends.

What is the representation of RNA species in the amplified population?
Our studies have consistently shown greater than 95% representation of RNA species in the amplified population when compared with unamplified RNA with starting concentrations greater than 100 ng.

When using a starting concentration of 5 ng of total RNA, some very low transcripts may be lost. Typically we obtain approximately 85% representation when starting with 5 ng.


siRNA Libraries

How do I retrieve my siRNA from the cell after selection?
Since each siRNA molecule is flanked by known sequences in the cloning vector, the siRNA inserts in the selected cells can easily be retrieved by PCR.

Do I have to use an Affymetrix microarray to identify the siRNA in the target cell population?
As an alternative to microarray analysis, the siRNA inserts could be recovered from clones by PCR, and identified by sequencing. However, microarray analysis provides the most efficient way to analyze enrichment of phenotype-associated siRNAs in a complex siRNA population.

How do I read the data after hybridization?
A CD with a gene/siRNA list and data analysis program, compatible with the Affymetrix data file, is provided with each siRNA library kit. This software enables the analysis of Affymetrix raw data and provides the sequences of siRNAs present in the GeneNet siRNA Library.

Can I buy individual clones from the GeneNet Library collection?
No. As opposed to collections of discrete siRNA sequences or cloned siRNA templates in 96-well plates, the GeneNet products are true cloned libraries in ready-to-use prepackaged VSV-G pseudotyped viral particles.

How can I verify the siRNA after screening if I cannot get the clone?
Although we do not provide individual clones from our library, specific clones can be obtained through the use of our pFIV siRNA Cloning and Expression Vectors once the siRNA inserts are identified, either by hybridization with the GeneChip microarray or by sequencing.

Ambion’s libraries contain validated siRNA, doesn’t that make them better?
Although marketed as validated, in actuality these validated siRNA only number a few hundred. The majority of the library is comprised of predicted siRNA and represents a much smaller set of genes.

It seems like this is just another way to do expression analysis with microarrays. Why it is better?
In some respect, the data is similar to an expression analysis. However, rather than just getting genes that are up and down regulated, this siRNA screening approach provides the investigator with genes, when inhibited, enabled their cells to survive selection or display the phenotype of interest. The genes targeted by the siRNA over-represented in the selected cell population are functionally involved in generating the phenotype or response of interest. This functional information is completely lacking with simple expression analysis.