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   Today: Last Update: 01 April 2013
 
 
   
 

Frequently Asked Questions

 

OpenBiosystems provide their own FAQ which can be accessed at:http://www.openbiosystems.com/Help/FAQ/

If you cannot find your answer here, please ask via email

How do i order shRNAi constructs from LISA?
Please compile a list of your genes of interest with at least their official symbol, species, and NCBI gene ID. The NCBI gene ID can be looked up at http://www.ncbi.nlm.nih.gov/gene. Please email this list (preferably as an Excel file) and the service you are interested in to lrf-orders <at> imb.uq.edu.au. We require the NCBI Entrez gene ID to uniquely identify your gene(s) of interest in the library annotation files. If appropriate you can state the species once for the entire set.

Has the knock-down efficacy of GIPZ-shRNAmir been validated?
No, generally speaking GIPZ-shRNAmir have been made in a random approach and have not been validated. However, since OpenBiosystems became part of Thermo Fisher Scientific a significant effort has been put into quality control, and Thermo Fisher has released a list of some 290 validated GIPZ-shRNAmir. The list can be found here or on the OpenBiosystems webpage.

Why are my glycerol stocks not growing?
E. coli
cells grown in microplates can settle and grow in the bottom of the well. It is advisable to thaw the glycerol stocks completely and mix the contents of each well before you use them for the first time. Once mixed, scraping cells from the top of a frozen culture will work.

Can i simply transfect the pGIPZ plasmid transiently?
Yes, the pGIPZ plasmid can be used to transiently knock-down gene expression if your cell line is susceptible to a transient transfection method.

Why does LISA use low-salt LB-Lennox bacterial growth medium?
The DNA cleavage activity of zeocin is inhibited by high ionic strength and non-neutral pH values. LB-Lennox contains only half the sodium chloride as standard LB. It is used to maintain the integrity of the library during long-term storage and replication.

Which antibiotic resistances does the pGIPZ plasmid confer to my cells?
When transforming or transfecting the entire pGIPZ plasmid into cells you will introduce resistance markers against carbenicillin / ampicillin, puromycin, zeocin, and hygromycin. When transducing cells with lentivirus produced from pGIPZ you will introduce the puromycin marker only.

What cell line are the pGIPZ plasmids actually stored in?
OpenBiosystems uses bacterial cultures of E. coli (Prime Plus) in LB-Lennox (5g NaCl / L) with 8% glycerol, 100μg/ml carbenicillin and 25μg/ml zeocin.

What cell line is E. coli Prime Plus?
PrimePlus are modified E. coli JM109 cells distributed by OpenBiosystems. Modification includes proprietary deletions that render these cells more stable than other competent cell lines, which, combined with stringent growth conditions will minimize recombination of lentiviral vectors and allow for greater quality plasmid DNA. Prime Plus cells should be grown with the appropriate antibiotic at 30°C for all incubations. If the growth period is to be followed by plasmid DNA isolation the cells can be grown at 37°C in order to increase DNA yield.

Why should subcloning only be done using an empty pGIPZ vector?
OpenBiosystems discourages the use of random pGIPZ constructs for cloning purposes:
This is because the restriction digests may not work completely and with an insert only ~345bp long you may not be able to tell the difference between which vector band still has a hairpin (if the digest didn't work to completion) and which band is actually an empty vector. Many colonies would have to be sequenced to confirm the presence of the new hairpin. Even if the empty pGIPZ vector is used to subclone into, sequencing one or two colonies is recommended. Also, there is a unique BamHI site in the stuffer of the empty vector that can be used as a diagnostic cut to determine if the clone still has the original stuffer, or if a hairpin has been cloned in. If the hairpin has been cloned in, no cut will be made.

I have the full hairpin sequence but not the sense sequence which aligns to the target gene.
The sense sequence can be obtained from the full hairpin sequence by using the Excel function “=MID(text, start position, length) as follows: =MID(full sequence, 21,19).

How can i look up shRNAmir sequences on the OpenBiosystems webpage?A step-by-step explanation is available as a pdf .

How should i grow my shRNAmir glycerols for amplification?
Within LISA we obtain the best results using standard LB broth with carbenicillin. Ampicillin can be used instead of carbenicillin. Zeocin should not be present. Zeocin and glycerol in the library storage medium interfere with subsequent DNA plasmid preparations and will reduce overall yield.

Can i transfer a pGIPZ insert into the inducible pTRIPZ?
Yes you can. For instance, you can use OpenBiosystems ‘Decode RNAi primers’ (PRM5038) to amplify the hairpin in the GIPZ vector and subclone it into the TRIPZ vector. More information is available in the TRIPZ manual.

What factors determine how well a lentivirus transduces a cell line?
Many factors determine how well a lentivirus transduces a cell line. These include host restriction factors, low nucleotide pools (resting cells), activity of the promoter, etc. GIPZ particles are pseudotyped with the VSV-G envelope and therefore gain access to the cell by binding to a lipid component of the plasma membrane, which is why VSV-G has such a wide tropism. The HIV envelope is not expressed in the vector, thus CD4/CXCR4/CCR5 is not utilized for entry.

What is hygromycin?
Hygromycin B is an antibiotic produced by the bacterium Streptomyces hygroscopicus. It is an aminocyclitol antibiotic with broad spectrum activity against prokaryotes and eukaryote through a dual effect on mRNA translation. Hygromycin B induces misreading of aminoacyl-tRNA by distorting the ribosomal A site (decoding center) and it affects the ribosomal translocation process.

How is the shRNA transcribed?
With pGIPZ plasmids, the turboGFP, puromycin, and shRNA are all transcribed as one long transcript. The shRNA ends up curling around on itself making the hairpin structure. Drosha then cleaves the hairpin off and turboGFP and puromycin are translated with the help of the IRES.

Can i use the hygromycin marker?
The hygromycin marker on both pGIPZ and pTRIPZ vectors was present on the parent plasmid they were constructed from but its function has never been tested by OpenBiosystems or LISA. Theorecically, the hygromycin marker can be used for selection of cells transfected with pGIPZ but hygromycin takes longer than puromycin to have the desired effect. Also, hygromycin selection will not work with transduced cells as the marker lies outside the LTRs of the vector and will not be packed into virus particles. The puromycin marker is part of the single GFP-shRNAmir transcript and will be part of the lentiviral genome.In pTRIPZ the hygromycin marker was specifically inactivated by OpenBiosystems and will not work.

What is zeocin?
Zeocin is an antibiotic from Invitrogen life technologies that is effective against bacteria, fungi (including yeast), plants and mammalian cell lines.
Zeocin is a formulation of phleomycin D1, a basic, water-soluble, copper-chelated glycopeptide isolated from Streptomyces verticillus. The copper-chelated form is inactive. When the antibiotic enters the cell, the copper cation is reduced from Cu2+ to Cu1+ and removed by sulfhydryl compounds in the cell. Upon removal of the copper, Zeocin is activated and will bind and cleave DNA, causing cell death. Zeocin resistance is conferred by the 13,665 Da protein product of the Sh ble gene which bindsZeocin stoichiometrically and inhibits its DNA strand cleavage activity. Expression of this protein in eukaryotic and prokaryotic hosts confers resistance to Zeocin.

Stable integration – linearize or not?
Linearization of the pGIPZ plasmid will improve integration into the mammalian genome but also reduce transfection efficiency to about 25%. If employed, linearization must be in an area that is inconsequential to those components that function post integration into the mammalian genome: if you linearize pGIPZ you should use FspI at base 10,755, which cuts inside the ampicillin marker.Remember though that each shRNA clone contains a unique target sequence as well as a unique barcode sequence of variable length. As a consequence, there is a small chance that any one clone could contain a second restriction site for the enzyme given. As a test, you can look up the sequence of the shRNAmir and the barcode on the OpenBiosystems webpage or digest a small aliquot of your DNA and run on a gel to ensure a single cut occurred.

What is the recommended pGIPZ sequencing primer?
The recommended pGIPZ sequencing primer is:
5'-GCATTAAAGCAGCGTATC-3'
The binding site lies from base 5820-5842 and runs in the reverse complement direction. The melting temperature of this 18-mer=52.7°C.

Can i make stable cell lines?
Yes. Determine the lowest concentration of puromycin that kills all your cells after 5-7 days. Transfect or transduce your cells with the shRNAmir of interest. Select cells with the experimentally determined concentration of puromycin 48 hours after transduction for about 1 week, regularly replacing the growth medium and washing away dead cells. Transfer individual surviving GFP-positive colonies into fresh vessels, re-grow to confluence under puromycin selection, then freeze as passage zero.

Why should i use lentivirus when i can simply transfect my assay cells?
A major advantage of the lentiviral delivery method is the ability to control the number of viral genomes that enter your target cells by adjusting the multiplicity of infection (MOI). In contrast, plasmid transfection results in the random and transient delivery of copy numbers that cannot be easily controlled.- when you are using the inducible pTRIPZ vector, a high copy number may magnify leakiness

Why should i titrate my virus?
Titration is essential to determining an accurate MOI in order to produce optimum results. Thus, it is critical to know the number of infectious units (IFU) in a given viral supernatant: use too little virus, and infections may be suboptimal; use excess virus, and cell performance may be compromised.

What is p24?
Recombinant lentivirus generated for research use, whether they are 3rd or 4th generation, contain major structural proteins including the VSV-G or ecotropic envelope proteins, matrix proteins, and virus core proteins. The gag gene encodes the viral capsid protein (p24); the nucleocapsid proteins (p6 and p7); and a matrix protein (p17).