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There have been 41 items by Functional Screens (Search limited from 17-July 18)
Open Biosystem, System Biosciences, GeneCopoeia, and Biosettia make lentiviral miRNA expression vector
Oirgene makes plasmid-based miRNA expression vector.
System Biosciences and Origene use dual promoters to express microRNA and selection marker, the problem is you might not know whether the miRNA is expressing when you selection marker is expressed from the other promoter. GeneCopoeia uses H1 promoter for miRNA expression and I guess they only express the pre-miRNA. I wonder the processibility of mature miRNA in the absence of flanking sequences in its precusor. Open Biosystem use one promoter and miRNA precursor is cloned in the "exon". I don't understand how the RFP and puro-resistant gene can be expressed while the same mRNA is processed by Drosha. Biosettia's miRNA expression vector has miRNA precursor cloned in the EF1a intron and RFP-puro expressed from the same promoter. I think it's a smart way to overexpress miRNA.
A lot of siRNA design was not compatible with Zamore rules, so I will suggest you to go to different online shRNA designer to see whether you can get one close to your siRNA sequence.
U6 promoter prefers G as start and H1 promoter prefers A or G (C or T should be fine). Your siRNA sequence starts from C, so you might want to use H1 promoter.
2. The primer sets you used for genome-typing are not good for RT-qPCR or qRT-PCR especially when you have genomic DNA contamination.
3. Dosage issue or shRNA potency; the same dosage works for your colleague because his target gene can be silenced in a lower concentration of shRNA. You might want to screen more potent shRNA by playing around the ratio of shRNA vector/luc-target vector (e.g. 1:1 or 1:5). Then use the shRNA for your in vivo experiments.
1. PRC primer set on the same exon
2. Genomic DNA contamination or gene homologue
3. Feedback regulation / compensation
4. cell culture condition (better change or split cells before assay)
I have not heard GFP expressed by U6 (pol III) promoter.
I've found a possible option on genecopeia but my supervisor pointed out that he has previously used plasmids in which the gfp expression is driven by U6 promoter whereas the plasmid that I found drives gfp expression under the cmv promoter. Because of this he said that our current packaging system may not work with this new plasmid.
Your packaging system has nothing to do with U6 promoter and GFP expression.
Depending on the lentiviral vectors, I found the following web site showing the differences between two general packaging systems.
My question is what part of the packaging system differs between the various companies and and as result determines whether a given plasmid will work with a particular packaging system?
I have used their lentiviral shRNA vector, The cloning strategy is very very clever and efficient.
You can also retrieve genomic sequence from UCSC database:
- Go to UCSC genome database
copy miRNA precursor sequence obtained from mirbase.org to blat genome.
it gives you 100bp flanking sequences on both 5' and 3' ends.
Non-homologous end-joining will join many copies of linearized vector and then homologous recombination will integrate multiple copies of your vector (the drug selection gene and shRNA expression cassette) into host genome. For example, a (unique) ScaI site in the Amp gene can be used to linearize the vector. Also, you might need to do longer selection to ensure the recombination/integration.
[size="2"]you said it's better to linearize the plasmid before transfection, If I understood properly, you mean cutting the plasmid with an enzyme, opening it then transfection.
It may be lethal especially in embryonic cells.
In your case, transfection efficiency matters. Linerization of vector is highly recommended. Longer selection time may be needed for vector integration.
By the way, is there any possibility that any splice variants have longer 3'UTR?
True, in my experiences using the 2nd generation packaging system seems not as good as using the 3rd generation system for 3rd generation lenti vector in virus production.
Second generation systems will also package third generation lentiviral vectors (in addition to second generation vectors).
This one should express TAT which is required for all second generation vectors and dispensible for third-gen
2. For non-viral vector, I have used pSUPER (OligoEngine), BLOCK-iT U6 and H1TO (Invitrogen), and pRNAi (Biosettia). Invitrogen and Biosettia RNAi vectors are linerized and OligoEngine's pSUPER is sold as circular plasmid. The linearized vectors give you much lower background (i.e. inoculate 2-3 colonies and you might get them all right). Personally I favor Biosettia's vector since only one DNA oligo is required to make shRNA clone, it's amazing and really high efficiency. I don't use next-door's free RNAi vectors any more because the oligo money I saved from using Biosettia's vector just like I get it for free. So, I don't bother to digest and gel-purify other RNAi vectors since then.
Below is the links for protocols:
3. The shRNA viral vector especially lentiviral shRNA vector overcomes the transfection issue since quite a few cells are hardly transfected. But the lentiviral packaging is a key, you better good on packaging. A lot of people use pLKO.1 since it's free but I am quite loyal to one oligo shRNA method to save time.
4. Linerize your non-viral shRNA vector before transfection, the non-homolog end joining and homologous recombination should give you multiple copy integration. check the protocols listed above.
Please note, for HIV/lenti infection: @12hr late-RT reaction reaches the peak; @24hr 2-LTR circle reaches the peak; @24hr integration reaches the peak. So, you are detecting most of late-RT products plus some LTR-circle and integrated cDNA. However, it gives you an idea how many copies of lentiviral genome get into your model cells.
Please wash your cells before extraction to remove plasmids carried over from your lentiviral transduction.