Viruses and cells
How to Generate Conditional Knockout Mice with Cre
The bacterial Cre recombinase targets a specific DNA sequence called loxP and deletes a segment of DNA flanked by loxP sequences. This system is often used in the generation of knockout and conditional knockout animals.
The knockout of specific genes leading to embryonic lethal phenotype will not yield adult animals. Cre-lox recombination provides a means to knockout the specific genes in adult mice, or to introduce a knockout phenotype in specific tissues (conditional knockout) using tissue-specific promoter driven Cre or an inducible Cre.
The cutting by Cre at the loxP sites and rejoining by ligase is an efficient process. During this process, inverted loxP sites will result in an inversion, whereas direct repeat will cause a deletion. Cre/lox recombination is a one-way reaction so there is no need for continued Cre expression. Therefore, Cre can be introduced by adenovirus or lenti/retrovirus. Here is an example of using adnovirus-Cre in one lab: for MEF, on a 70% confluent P10 cm plate (probably 2-2.5 million cells), use 6ul of 1.1×10^12 adenovirus-Cre, which will give 80% infection; or use 10ul of 1.1×10^12 adenovirus-Cre to get 90% infection, with GFP as marker and analyzed by FACS.
Adenovirus could post a toxicity problem when used at very high titers to reach high percentage of transduction. An alternative is to use only lentivirus-Cre, at only about 1-2 ul and still obtain >80% infection. However, a silencing event needs to occur before the expression of Cre from lentivirus is shut off. The timing and degree of silencing is not controlled in such experiments. Continued expression of Cre should not influence most experiments.
To be certain that the Cre enzyme can be successfully delivered into the nucleus for conditional knockout to occur, the bacterial Cre gene needs to be engineered to contain a nuclear localization (nl) signal of eukaryotic cells. The function of the nuclear-localized Cre (nlCre) can be tested using a loxP-nuclear localized lacZ (nlacZ) reporter cell line, which can be used to monitor the function of the nlCre recombinase.
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Choosing siRNA, shRNA, and miRNA for Gene Silencing
RNAi refers to dsRNA-induced gene silencing, a cellular process that degrades RNA homologous to one strand of the dsRNA [1, 2]. The intermediates of long dsRNA-initiated RNAi are double-stranded small interfering RNAs (siRNA), typically 21-23 nucleotide (nt) long. The siRNAs, when introduced into cells, can be used to silence genes in mammalian systems where long dsRNAs prompt protein kinase R (PKR), RNase L, and interferon activities that result in non-specific RNA degradation and general shutdown of protein synthesis [3]. siRNAs can either be chemically synthesized then directly transfected into cells or can be generated inside the cell by introducing vectors that express short-hairpin RNA (shRNA) precursors of siRNAs. The process of shRNA into functional siRNA involves cellular RNAi machinery that naturally process genome encoded microRNAs (miRNA) that are responsible for cellular regulation of gene expression by modulating mRNA stability, translation, and chromatin structures [4].
Chemically synthesized siRNA is the simplest format for RNAi. One of the biggest hurdles for achieving effective RNAi with siRNA is that many cells are difficult to transfect. An RNAi experiment is typically considered successful when the target gene expression is reduced by >70%, a threshold not reachable by many types of cells due to their low transfection efficiency. Another drawback of using synthetic siRNA is the limited duration of post-transfection effects, typically with gene silencing activities peaking around 24 hours, and diminishing within 48 hours [5]. Chemical synthesis of siRNA, which is a service Allele Biotech and Orbigen (now merged under the Allele brand) pioneered and still provides, is expensive on a per transfection basis relative to DNA vector based reagents.
shRNA can be introduced by DNA plasmid, linear template, or packaged retroviral/lentiviral vectors. Using any form of DNA construct, except the PCR template format such as Allele’s LineSilence platform, requires creating DNA constructs and sequence verification; a taxing work load if multiple genes need to be studied. However, once the constructs are made, they can be reproduced easily and inexpensively. It is difficult to directly compare the effectiveness of siRNA versus shRNA on a per molecule basis because RNA polymerase III (Pol III) promoters such as U6 or H1 commonly used to express shRNAs can make thousands of copies of shRNA from a single DNA template. However when both siRNA and shRNA are produced the same way, e.g. synthesized chemically, shRNA is reported to be somewhat more effective [6, 7]. For the goals of this research, the most important advantage using shRNA can provide over siRNA is that it can be carried on a lentiviral vector and introduced into a wide variety of cells.
Similar to the comparison between siRNA versus shRNA, it is also difficult to rank the efficiency of shRNA versus miRNA from published data, partly due to different results from different experimental systems. There have been several reports that showed shRNA can cause significant cell toxicity, especially in vivo such as after injection into mouse brain. It was originally reasoned that highly efficient expression from Pol III promoters might overwhelm the cellular machinery that is needed to execute endogenous RNAi functions such as transporting miRNA from the nucleus to the cytoplasm. It was later found out that even using Pol III promoter to create miRNA could still mitigate the toxic effects of shRNA [8]. Since shRNA and miRNA are processed by endonuclease Dicer before being incorporated into RNA induced silencing complex (RISC), the exact identity of siRNAs produced from a given shRNA or miRNA targeting the same region on the mRNA are not known in most of the earlier studies. By designing shRNA and miRNA to give exactly the same processed siRNAs, Boudreau et al. showed that shRNA is actually more potent than miRNA in various systems [9].
New Product/Service of the Week (02-01-10 to 02-07-10): Lentrivirus retrovirus shRNA Packaging Services as low as under $900 per virus.
Promotion of the week: Get mouse tail lysis buffer, human blood genotyping buffer, or DNA purification kit, and get Allele Biotech’s superior PCR MasterMix for free.
1. Fire, A., S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, and C.C. Mello, Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998. 391(6669): p. 806-11.
2. Hannon, G.J., RNA interference. Nature, 2002. 418(6894): p. 244-51.
3. McManus, M.T. and P.A. Sharp, Gene silencing in mammals by small interfering RNAs. Nat Rev Genet, 2002. 3(10): p. 737-47.
4. Hutvagner, G. and P.D. Zamore, A microRNA in a multiple-turnover RNAi enzyme complex. Science, 2002. 297(5589): p. 2056-60.
5. Rao, D.D., J.S. Vorhies, N. Senzer, and J. Nemunaitis, siRNA vs. shRNA: similarities and differences. Adv Drug Deliv Rev, 2009. 61(9): p. 746-59.
6. Vlassov, A.V., B. Korba, K. Farrar, S. Mukerjee, A.A. Seyhan, H. Ilves, R.L. Kaspar, D. Leake, S.A. Kazakov, and B.H. Johnston, shRNAs targeting hepatitis C: effects of sequence and structural features, and comparision with siRNA. Oligonucleotides, 2007. 17(2): p. 223-36.
7. Siolas, D., C. Lerner, J. Burchard, W. Ge, P.S. Linsley, P.J. Paddison, G.J. Hannon, and M.A. Cleary, Synthetic shRNAs as potent RNAi triggers. Nat Biotechnol, 2005. 23(2): p. 227-31.
8. McBride, J.L., R.L. Boudreau, S.Q. Harper, P.D. Staber, A.M. Monteys, I. Martins, B.L. Gilmore, H. Burstein, R.W. Peluso, B. Polisky, B.J. Carter, and B.L. Davidson, Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi. Proc Natl Acad Sci U S A, 2008. 105(15): p. 5868-73.
9. Boudreau, R.L., A.M. Monteys, and B.L. Davidson, Minimizing variables among hairpin-based RNAi vectors reveals the potency of shRNAs. Rna, 2008. 14(9): p. 1834-44.
Commonly Known Facts About Viral Packaging -That Might Not Be Correct…
Packaging lentiviruses or retroviruses is not a routine procedure that every biology lab performs even if there is need to use it. A viral packaging protocol normally begins with preparation of purified transfer plasmid DNA, a miniprep should be enough for a few transfections. The virus backbone plasmid is either co-transfected into commonly used cells with helper plasmids that provide the essential proteins required for particle packaging, or transfected into established helper cell lines that express the required proteins from integrated transgenes. After incubation of packaging cells for a couple of days, viruses are collected and tittered. Titer determination is somewhat tricky for the inexperienced. Using a control virus expressing a fluorescent protein can make this step convenient.
Commonly known facts:
1) Lentiviruses are packaged at a titer of 10^6 IU/ml without concentrating steps.
This needs update since with more advanced technologies lentiviruses can be packaged routinely at 10^8 IU/ml. With further concentrating, the titer can be easily above 10^11 IU/ml. Retroviruses can be packaged to similar titers as well.
2) Using packaging cell lines gives the highest possible titer
While packaging cell lines (such as Allele’s popular Phoenix Eco and Ampho cells for retrovirus packaging) provides maybe the most convenient method for packaging, the yield will not reach the highest potential. Packaging cell lines may also lose their capability for packaging after continued culturing, requiring periodic selection with antibiotics and functional tests, as we do here at Allele.
3) Retroviruses are always collected in one shot after transfection into packaging cells
If the transfer vector has oriP/EBNA1 episomal maintenance system, such as some of the Phoenix vectors Allele offers, the plasmids may continue to express for up to 30 days. With puromycin selection, the titer of retrovirus produced from Eco or Ampho cells can reach 10^7 IU/ml.
This week’s promotion (102509-103109): 10% off across the board of Allele Biotech’s custom services, for an example, check out our world-leading baculovirus protein expression.
New Product/Service of the Week: Introduction of Custom Viral Packaging Service. Routine titer of 10^8 IU/ml, as high as 10^10 IU/ml, option to include cloning. Signature service ABP-CS-MERV002 provides more than 200 million particles at $7/million particles. These are game-changing prices for the viral packaging service market based on superior technologies!
Introducing Baculo Virus Expression System (BVES) with a Strong IRES
Internal ribosome entry site (IRES) can be used to initiate translation of a second open reading frame (ORF) of an mRNA, providing the benefits of: 1) avoiding promoter competition in a dual promoter situation; 2) having controlled ratio of expression of two proteins; 3) placing a dominant selection pressure on the entire bicistronic mRNA and hence the maintenance of the transgene when a selection marker is placed as the second ORF.
IRES elements are located mainly in RNA viruses except certain mammalian and insect mRNA molecules. Only one DNA virus has so far been found to contain an IRES, the while spot syndrome virus (WSSV) of marine shrimp. This IRES, compared to a very few other choices known to function in insect cells such as the IRES from Rhopalosiphum padi virus (RhPV), has strong translation initiation activity (~98-99% in reference to cap-dependent initiation), insect cell specificity, and encompasses only 180 base pairs.
Allele Biotech, with its acquisition of Orbigen, is a major provider of BVES products and services with more than 10 years of experience. Allele’s featured New Products of the Week* this week are WSSV IRES containing baculovirus vectors, the sIRES (for Strong IRES from Shrimp virus) series plasmids. Currently one version is pOrb-MCS-sIRES-VSVG for pseudotyping baculoviruses (within the Emerald Baculovirus for Mammalian Expression series), with pOrb-mWasabi-sIRES-VSVG as a fluorescent protein control; the other is pOrb-MCS-sIRES-MCS for cloning a custom second cDNA. New versions in the future will include IRES driven mWasabi and other commonly used selection markers.
With a current research project for the National Cancer Institute (NCI) within the National Institutes of Health (NIH) involving development of modified BVES and mammalian protein expression and purification systems, Allele Biotech expects this product line to continue its expansion at a fast pace.
* Allele Biotech announces at least one new product every Wednesday through news release at AlleleNews or Allele Blog and social networks.
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