|AX0010007||1.0 µg DNA|
This vector contains the human REST gene (N-term GST and FLAG tagged) cloned into pGEX-KG using BamHI/XhoI. This vector was submitted to the abmXchange program by Yan Quan Zhang (University of Kentucky).
|Unit quantity||1.0 µg DNA|
|Alternate Accession Numbers|
|Full Gene Name||RE1 silencing transcription factor|
Cloned into pGEX-KG vector by BamHI/XhoI
|Storage Buffer||10mM Tris-HCI, 1mM EDTA, pH8.0|
|Insert Sequence|| 1 accatggatt acaaggatga cgacgataag atcccagtta tggccaccca ggtaatgggg |
61 cagtcttctg gaggaggagg gctgtttacc agcagtggca acattggaat ggccctgcct
121 aacgacatgt atgacttgca tgacctttcc aaagctgaac tggccgcacc tcagcttatt
181 atgctggcaa atgtggcctt aactggggaa gtaaatggca gctgctgtga ttacctggtc
241 ggtgaagaaa gacagatggc agaactgatg ccggttgggg ataacaactt ttcagatagt
301 gaagaaggag aaggacttga agagtctgct gatataaaag gtgaacctca tggactggaa
361 aacatggaac tgagaagttt ggaactcagc gtcgtagaac ctcagcctgt atttgaggca
421 tcaggtgctc cagatattta cagttcaaat aaagatcttc cccctgaaac acctggagcg
481 gaggacaaag gcaagagctc gaagaccaaa ccctttcgct gtaagccatg ccaatatgaa
541 gcagaatctg aagaacagtt tgtgcatcac atcagagttc acagtgctaa gaaatttttt
601 gtggaagaga gtgcagagaa gcaggcaaaa gccagggaat ctggctcttc cactgcagaa
661 gagggagatt tctccaaggg ccccattcgc tgtgaccgct gcggctacaa tactaatcga
721 tatgatcact atacagcaca cctgaaacac cacaccagag ctggggataa tgagcgagtc
781 tacaagtgta tcatttgcac atacacaaca gtgagcgagt atcactggag gaaacattta
841 agaaaccatt ttccaaggaa agtatacaca tgtggaaaat gcaactattt ttcagacaga
901 aaaaacaatt atgttcagca tgttagaact catacaggag aacgcccata taaatgtgaa
961 ctttgtcctt actcaagttc tcagaagact catctaacta gacatatgcg tactcattca
1021 ggtgagaagc catttaaatg tgatcagtgc agttatgtgg cctctaatca acatgaagta
1081 acccgccatg caagacaggt tcacaatggg cctaaacctc ttaattgccc acactgtgat
1141 tacaaaacag cagatagaag caacttcaaa aaacatgtag agctacatgt gaacccacgg
1201 cagttcaatt gccctgtatg tgactatgca gcttccaaga agtgtaatct acagtatcac
1261 ttcaaatcta agcatcctac ttgtcctaat aaaacaatgg atgtctcaaa agtgaaacta
1321 aagaaaacca aaaaacgaga ggctgacttg cctgataata ttaccaatga aaaaacagaa
1381 atagaacaaa caaaaataaa aggggatgtg gctggaaaga aaaatgaaaa gtccgtcaaa
1441 gcagagaaaa gagatgtctc aaaagagaaa aagccttcta ataatgtgtc agtgatccag
1501 gtgactacca gaactcgaaa atcagtaaca gaggtgaaag agatggatgt gcatacagga
1561 agcaattcag aaaaattcag taaaactaag aaaagcaaaa ggaagctgga agttgacagc
1621 cattctttac atggtcctgt gaatgatgag gaatcttcaa caaaaaagaa aaagaaggta
1681 gaaagcaaat ccaaaaataa tagtcaggaa gtgccaaagg gtgacagcaa agtggaggag
1741 aataaaaagc aaaatacttg catgaaaaaa agtacaaaga agaaaactct gaaaaataaa
1801 tcaagtaaga aaagcagtaa gcctcctcag aaggaacctg ttgagaaggg atctgctcag
1861 atggaccctc ctcagatggg gcctgctccc acagaggcgg ttcagaaggg gcccgttcag
1921 gtggagccgc cacctcccat ggagcatgct cagatggagg gtgcccagat acggcctgct
1981 cctgacgagc ctgttcagat ggaggtggtt caggaggggc ctgctcagaa ggagctgctg
2041 cctcccgtgg agcctgctca gatggtgggt gcccaaattg tacttgctca catggagctg
2101 cctcctccca tggagactgc tcagacggag gttgcccaaa tggggcctgc tcccatggaa
2161 cctgctcaga tggaggttgc ccaggtagaa tctgctccca tgcaggtggt ccagaaggag
2221 cctgttcaga tggagctgtc tcctcccatg gaggtggtcc agaaggagcc tgttcagata
2281 gagctgtctc ctcccatgga ggtggtccag aaggaacctg ttaagataga gctgtctcct
2341 cccatagagg tggtccagaa ggagcctgtt cagatggagt tgtctcctcc catgggggtg
2401 gttcagaagg agcctgctca gagggagcca cctcctccca gagagcctcc ccttcacatg
2461 gagccaattt ccaaaaagcc tcctctccga aaagataaaa aggaaaagtc taacatgcag
2521 agtgaaaggg cacggaagga gcaagtcctt attgaagttg gcttagtgcc tgttaaagat
2581 agctggcttc taaaggaaag tgtaagcaca gaggatctct caccaccatc accaccactg
2641 ccaaaggaaa atttaagaga agaggcatca ggagaccaaa aattactcaa cacaggtgaa
2701 ggaaataaag aagcccctct tcagaaagta ggagcagaag aggcagatga gagcctacct
2761 ggtcttgctg ctaatatcaa cgaatctacc catatttcat cctctggaca aaacttgaat
2821 acgccagagg gtgaaacttt aaatggtaaa catcagactg acagtatagt ttgtgaaatg
2881 aaaatggaca ctgatcagaa cacaagagag aatctcactg gtataaattc aacagttgaa
2941 gaaccagttt caccaatgct tcccccttca gcagtagaag aacgtgaagc agtgtccaaa
3001 actgcactgg catcacctcc tgctacaatg gcagcaaatg agtctcagga aattgatgaa
3061 gatgaaggca tccacagcca tgaaggaagt gacctaagtg acaacatgtc agagggtagt
3121 gatgattctg gattgcatgg ggctcggcca gttccacaag aatctagcag aaaaaatgca
3181 aaggaagcct tggcagtcaa agcggctaag ggagattttg tttgtatctt ctgtgatcgt
3241 tctttcagaa agggaaaaga ttacagcaaa cacctcaatc gccatttggt taatgtgtac
3301 tatcttgaag aagcagctca agggcaggag taatgaaact ttgaacaagg tttcagttct
3361 tagtttgtaa ggtatattac attttatatt catttatgat agcagacaac cttttaagat
3421 tgctttaatt agtatctgat gttgattttt aagtggcatt cttttcctta ggacttttta
3481 tgtatacctg ttgattgttg tgtaaatttt agtaaatcta agagagtgta ctaaaccagc
3541 aggtatctgt tagcttatgt gtttaattga aattagaagg ctaagatggt ataacagcat
3601 tttattgctt tgtccagcta caacatgtca tttttttctc catgtcttat cttcctgttt
3661 cactttagtt tattcttcgt tttttattga gatccactag taacggccgc cagtgtgctg
3721 gaattttgca gatatccatc acactggcgg ccg
1 year when stored at -20°C or lower in a non-frost free freezer.
|Disclaimer||1) Disclaimer for Transcript Variants: The provided accession number refers to the transcript (mRNA) sequence for this product. The molecular sequence of this clone aligns with the gene accession number as a point of reference only. However, individual transcript sequences of the same gene can differ through naturally occurring variations (e.g. polymorphisms), each with its own valid existence. This clone is substantially in agreement with the reference, but a complete review of all prevailing variants is recommended prior to use. All sales are final.|
2) Disclaimer for Gene Sequence: The provided accession number refers to the transcript (mRNA) sequence for this product. Please verify that this is the desired transcript sequence by cross-referencing. This is important because a single gene can have multiple different transcripts owing to naturally occurring variations. All sales are final.
3) Disclaimer for Intended Use: All of abm's vectors and viral particles are for research use ONLY and NOT for therapeutic/diagnostic applications. abm is not liable for any repercussions arising from the use of its vector(s) in therapeutic/diagnostic application(s).
4) Disclaimer for Extra Nucleotides: Cloning may lead to the insertion of extra nucleotides at the 5' or 3' end of the target sequence which, in most cases, is innocuous to the stability/functionality of the construct.
5) abm guarantees that at least 1 out of the 3 sgRNA constructs purchased in a set designed to be used with Cas9 Nuclease will result in gene knock-out due to frameshift mutations in over 50% of cells, after successful infection and drug selection. This guarantee applies to sgRNAs designed to target human, mouse or rat genes only. If knock-out is not achieved in extremely rare cases, a one-time replacement of another set of 3 targets with alternative sgRNA sequences will be provided. To qualify for this replacement, customers must examine knock-out efficiency by Surveyor assay. Before sending your inquiry, please make sure you have optimized your experiments as far as possible. This includes (where applicable) increasing and optimizing your MOI, increasing the duration of infection (up to 72 h), and carrying out clone screening before assaying for knock-out. Please also provide data to show that a reporter virus was used to optimize the MOI for your target cell line. Customers must provide adequate data to show >80% infection efficiency with a positive control, plus additional qPCR data to evaluate the level of mRNA expression.
For vector transfection, please evaluate the vector transfection efficiency by detecting Cas9 or puromycin expression for the 'All-in-One' vectors using qPCR, or neomycin for constructs containing only the sgRNA. In addition, please provide Surveyor Assay or Sanger Sequencing data on at least 20 isolated clones.
abm limits its obligation and liability for the success of this technology to providing one replacement of any sgRNA lentivector product only. The replacement set will not be covered by the same guarantee. If these constructs are also considered to be ineffective then the gene is most likely not susceptible to sgRNA knock-out.
|Why use a retroviral expression system?|
Expression systems based on retroviral gene delivery are generally more reliable and have broader utility than standard plasmid-based transfection systems. Retroviral gene delivery (infection) is usually used instead of transfection when cells are difficult to transfect. Retroviruses preferentially integrate into actively transcribed regions of the genome. The infection yields a cell line that stably expresses your gene of interest.
|What is the limit on the size of my DNA insert?|
Retroviruses efficiently package RNA that is approximately 8-9 kb. Most developed vectors can take as large as ~6 kb and still be packaged with no reduction in viral titer.
|Which cells are recommended to produce the retrovirus?|
Phoenix A cells are recommended for retrovirus.
|Do you have any further information about the Retro-combo mix (E-510) and the pack easy cells (E-511)?|
The Retro-combo mix is a two plasmid mixture of retro Gag/poly and VSVG. The packaging cells are a 293 derivative optimized for retrovirus production.
|I cannot detect expression for my gene of interest. Why?|
Retroviral vector transduction efficiency is very low and can only intergrate a single copy into infected target cells (for most cell types). Thus, we recommend transfection with your vector in 293 cells for a fusion gene expression assay before your viral production and infection. If your insert is in frame, the sequence is correct and there is still no signal after transfection in 293 cells, there may be technical difficulties with the western blot. Make sure the HA or His antibody is of high quality. Our Tag antibodies are carefully produced to handle most fusion vectors that are not detectable with Tag antibodies from other sources. We also have HA and His cell lysates for a true positive control. It is important to note that HA or His tag antigenecity may be altered by adjacent amino acids close to the HA or His tag. This is why some companies specify that you need a specific amino acid upstream or downstream of HA or His tags. (For example, adding an Ala before the His tag). Based on our years of experience with Tag antibodies, there are about 10% of vectors with correct fusion genes that are not detectable even by excellent antibodies.
|What is the reason for putting the SV40 promoter region after the multiclonal site?|
For retroviral vector, 5'-LTR has been traditionally used as the default promoter which will also override any down-stream promoters. If you visit other suppliers’ websites, there are quite a few retroviral vectors using 5'-LTR promoter. Later studies show that the CMV promoter down stream of 5'-LTR does not work. So both promoters are fine. Promoter is less of an issue here.
|Can I freeze the viral supernatant and use it later?|
Yes. Collect the virus at 48 hours after transfection, filter with a 0.45µm cellulose acetate or polysulfonic filter, and then freeze at –80°C. You do not need to add a cryopreservative such as glycerol or DMSO. When you are ready to use the frozen supernatant, thaw it quickly by placing it in a 37°C water bath until ice crystals have just disappeared.
|Is the poly A + sequence needed when cloning my gene of interest into a retroviral vector?|
You do not need to include the poly A + sequence with your gene because the native polyadenylation signal (from the wild-type virus) that is located in the 3' LTR of most retroviral vectors is sufficient for polyadenylation of the transcribed gene of interest.
|What is the safety concerns associated with using retroviral vectors and packaging cells?|
The National Institute of Health and the Center for Disease Control have designated retroviral vectors such as those from Moloney murine leukemia virus (MoMuLV) as Biosafety Level 2. NIH guidelines require that retroviral production and infection be performed in a Biosafety Level 2 facility. Recombinant retroviruses are extremely labile and are inactivated by ethanol, detergents, or bleach Because of their lipid-derived membrane.
|What is the difference between Retro-, Lenti-, and Adeno- viruses?|
Retrovirus: Classic, can integrate into the genome but with low transduction efficiency. They are useful for gene transfer and protein expression in cells that have low transfection efficiency with other transfection reagents. Lentivirus: Can integrate into the genome with relatively high transduction efficiency and they are very useful for cells that have low transfection efficiency with other transfection reagents. No special competent cells required, as they are stable plasmids. Lentiviruses are a powerful tool for stable gene transfer to both dividing and non-dividing cells in vitro and in vivo. Adenovirus: Only work transiently (about 7 days) but have almost 100% transduction efficiency. Adenoviruses can infect a broad range of cell types with the highest efficiency and infection is not dependent on active host cell division. A second key feature is that high virus titers and high-level gene expression can be obtained in most mammalian cells.
|What are the correct concentration units for each recombinant viral particle?|
For lentiviruses and retroviruses, they are measured in CFU/ml (colony-forming units per millilitre). Transduction with lentiviruses and retroviruses can cause the formation of colonies, which can be quantified for concentration. For AAV the titer is measured as genome copies per mL (GC/mL). Adenoviruses are measured as PFU/ml (plaque-forming units per millilitre). Transduction with adenoviruses will kill packaging cells, forming plaques in the process for quantification. The concentration for all three types of viruses can also be classified as IU/ml (Infectious Units/ml). Ultimately, the units refers to the viral particles and different units reflect the different assays involved.
|How long after transduction can the infection efficiency be observed?|
You can observe transduction efficiency from 48 hours up to 5 days after infection.
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