All pre-made retrovirus controls are reconstituted in 0.1 ml, 10% glycerol + PBS, in ready-to-use format.
| Product | Cat. # | Gene | Promoter | Titer | Size | Price | |
| Retro-Null | R1000 | Null | CMV | 1x10e12 lp/ml | 0.1 ml | $500.00 | |
| Retro-GFP | R1020 | EGFP | CMV | 1x10e12 lp/ml | 0.1 ml | $500.00 | |
| Retro-shControl | R1040 | Scramble shRNA | U6 | 1x10e12 lp/ml | 0.1 ml | $500.00 | |
| Retro-shControl-GFP | R1050 | Scramble shRNA, GFP co-expression | U6, CMV | 1x10e12 lp/ml | 0.1 ml | $500.00 |
Welgen provides a customer 3 ml of 1×1012 viral particles/ml in our standard amplification/purification services (CAT#3000). If a customer needs less amount of adenoviral stock, Welgen also can do it for you. Existing adenovirus vectors supplied by our customers or newly constructed vectors will be amplified to 1 x 1012 viral particles. The adenovirus will then be purified by centrifugation using two sequential cesium chloride gradients. The final product (1 ml, 1×1012 viral particles/ml) will be dialyzed, tittered spectrophotometrically, and tested for sterility, and then shipped to customer. The service time is approximately 2 weeks.
Existing adenovirus vectors supplied by our customers or newly constructed vectors will be amplified to obtain more than 3 x 1012 viral particles, which requires approximately 2 weeks. The adenovirus will then be purified by centrifugation using two sequential cesium chloride gradients. The final product (3 ml, 1×1012 viral particles/ml) will be dialyzed, tittered spectrophotometrically, and tested for sterility, and then shipped to customer.
Three short hairpin RNA (shRNA) sequences will be selected and cloned into a pQuietU6 plasmid. The desired constructs will be sequenced to verify the correct shRNA. The 3 shRNA plasmids will then be co-transfected into 293 cells with an cDNA expression plasmid. The most effective shRNA can be determined by a Western blot. Welgen guarantees that the selected vector will provide 70% silencing efficiency in a co-transfection experiment.
The customer will provide Welgen an effective shRNA sequence. Welgen will be synthesized and inserted it into pQuietU6 vector and convert it into an adenovirus. A crude cell lysate (106 vp/ml) can be shipped to customer within 2 weeks. The crude cell lysate is suitable for testing to ensure that the virus performs as expected. Additional amplification and purification of the virus (Cat#S3000) will require another 2 weeks.
The customer will provide us a validated shRNA (short hairpin RNAi) sequence. Welegn will synthesize a double-stranded oligonucleotide that encodes the validated shRNA sequence and inserted into a pQuiet vector. The vector will be amplified and sequenced to ensure that the correct shRNA sequence will be expressed. You will receive your plasmid within 2 weeks. If you would like more than one sequence to be inserted into pQuiet 2, 3, or 4 for multiple gene silencing, the additional cost will be charged per sequence. All you need to do is to provide the validated, effective 19 bp sequences from the target genes.
The multiplicity of infection or MOI is the ratio defined by the number of infectious virus particles divided by the number of target cells. In this service, MOI will be assayed using cells (provided by customer or by Welgen). It usually takes two to three weeks.
More than 5 ug high quality adenoviral DNA will be purified from approximately 1011viral particles.
HEK293 cells growing on 96 well plates will be infected with serial dilutions of an adenoviral vector stock and the resulting infection pattern will be evaluated to determine the TCID50 (Tissue Culture Infectious Dose 50) using the Karber formula.
The standard method to determine the presence of RCA (Replication-Competent Adenovirus) is to inoculate A549 cells with a sample of the viral stock. Since A549 cells are very readily infected by adenovirus but do not express the E1 gene, no plaque will form unless viral particles that have acquired the E1 gene are present. The sensitivity of this method is limited by the cytotoxic effects of the replication-deficient virus on the A549 cells, which limits the number of particles that can be added to the cultured cells. Approximately 1 RCA per 106 viral particles (determined spectrophotometrically) can be detected by this method.
The DNA purified from viral particles is used as a template for a PCR procedure in which E1 gene specific primers are used to detect the presence of the E1 gene. The sensitivity of this method is limited by the amount of template DNA that can be added. Greater sensitivity (10-fold) can be achieved by doing 10 replicate PCR reactions. The PCR procedure to detect the presence of RCA is faster and more sensitive than the assay that employs A549 cells and is recommended before amplification of a viral stock.
The abundance of adenoviral particles can be determined in a variety of ways. Two of the most useful are the spectrophotometric method, which is fast and highly reproducible, and the plaque assay, which estimates the number of infectious particles that are present. The plaque assay is performed by treating host cells in which the virus can replicate with serial dilutions of the virus preparation for several days. During the incubation, infected cells grow new viral particles that infect neighboring cells, which die and release more adenovirus until a plaque becomes visible. The plaque assay is slower and less reproducible than the spectrophotometric assay, but it provides a result that may be a more realistic estimate of the number of viral particles needed to evoke a biological response. A normal ratio between the spectrophotometric and plaque assay results indicates that a purified viral product is not contaminated with UV absorbent material.
Newly generated vectors that have been constructed using our adenoviral system do not require plaque purification, but when previously amplified vectors are re-amplified, rare crossover events can result in the appearance of RCA (Replication-Competent Adenovirus), which must then be eliminated by plaque purification. Recombination is expected to occur only after adenovirus has been amplified extensively and generally results in replacement of the desired transgene with the E1 gene that was deleted to make the virus replication-deficient. Amplification of viral stocks contaminated by RCA results in overgrowth by the RCA. It is always prudent to perform an RCA assay (#S3130 or S3140) before re-amplifying a viral stock.
Existing adenovirus vectors supplied by our customers or newly constructed vectors will be amplified to obtain more than 3 x 1012 viral particles, which requires approximately 2 weeks. The adenovirus will then be purified by centrifugation using two sequential cesium chloride gradients. The final product (3 ml, 1×1012 viral particles/ml) will be dialyzed, tittered spectrophotometrically, and tested for sterility, and then shipped to customer.
Welgen provides a customer 3 ml of 1×1012 viral particles/ml in our standard amplification/purification services (CAT#3000). If a customer needs less amount of adenoviral stock, Welgen also can do it for you. Existing adenovirus vectors supplied by our customers or newly constructed vectors will be amplified to 1 x 1012 viral particles. The adenovirus will then be purified by centrifugation using two sequential cesium chloride gradients. The final product (1 ml, 1×1012 viral particles/ml) will be dialyzed, tittered spectrophotometrically, and tested for sterility, and then shipped to customer. The service time is approximately 2 weeks.
By using this service, the customer provides the adenovirus construct and Welgen will generate adenovirus in 293 cells. Welgen will deliver a crude cell lysate containing the viral construct (approximately 106 viral particles/ml) within 2 weeks. The crude cell lysate is suitable for testing to ensure that the virus performs as expected. Additional amplification and purification of the virus (Cat#S3000) will require another 2 weeks.
The customer will provide Welgen an effective shRNA sequence. Welgen will be synthesized and inserted it into pQuietU6 vector and convert it into an adenovirus. A crude cell lysate (106 vp/ml) can be shipped to customer within 2 weeks. The crude cell lysate is suitable for testing to ensure that the virus performs as expected. Additional amplification and purification of the virus (Cat#S3000) will require another 2 weeks.
The customer will provide Welgen an effective shRNA sequence. Welgen will be synthesized and inserted it into pQuietU6 vector and convert it into an adenovirus. A crude cell lysate (106 vp/ml) can be shipped to customer within 2 weeks. The crude cell lysate is suitable for testing to ensure that the virus performs as expected. Additional amplification and purification of the virus (Cat#S3000) will require another 2 weeks.
The customer will provide us a validated shRNA (short hairpin RNAi) sequence. Welgen will synthesize a double-stranded oligonucleotide that encodes the validated shRNA sequence and inserted into a pQuiet vector. The vector will be amplified and sequenced to ensure that the correct shRNA sequence will be expressed. You will receive your plasmid within 2 weeks. If you would like more than one sequence to be inserted into pQuiet 2, 3, or 4 for multiple gene silencing, the additional cost will be charged per sequence. All you need to do is to provide the validated, effective 19 bp sequences from the target genes.
For this service:
Required Materials:
Turnaround Time:
Deliverables:
Final viral yield may depend on the nature of transgene.
For this service:
Required Materials:
Turnaround Time:
Deliverables:
If a customer does not provide the cDNA, Welgen will clone the cDNA or purchase the cDNA for the customer. The cDNA will be inserted into our adenovirus system and generate adenovirus. A crude cell lysate containing the viral construct (approximately 106 viral particles/ml) can be delivered to the customer within 3 weeks. The crude cell lysate is suitable for testing to ensure that the virus performs as expected. Additional amplification and purification of the virus (Cat# S3000) will require another 2 weeks.
Welgen will use insert your sequence into the adenoviral genome and return a crude cell lysate containing the viral construct (approximately 109 viral particles/ml) within 2 weeks. The crude cell lysate is suitable for testing to ensure that the virus performs as expected. Additional amplification and purification of the virus (Cat#S3000) will require another 2 weeks. Typically we will need about 10 ug of a plasmid containing the gene to be expressed.