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trigger of RNAi gene silencing response in mammalian cell culture as well as animal model systems. We offer a Lentivirus and a Murine Stem Cell Virus (MSCV)
derived retrovirus system for expressing shRNA in vivo. Both systems feature a human U6 snRNA gene promoter or a H1 RNA gene promoter for expressing shRNA
hairpin by RNA polymerase III, which initiates from defined transcription start site and recognizes a run of "T" for termination. The U6 promoter has a stringent
requirement of a "G" nucleotide as the initiator sequence whereas the H1 promoter accepts "G, A, or C". So, when designing shRNA target sequence for use in the
U6 transcription, it is preferable to start with a "G". However, some researchers also add a "G" to those proven target sequences that do not begin with "G". Both of
these viral expression systems carry a GFP marker for in vivo identification, and the MSCV based system also carry a puromycin selection marker. These constructs
can be used either as plasmid DNA for transient transfection into your cell lines or alternatively they can be assembled into viral particles for use in infecting cells.
For Design & Construction service, we only need your gene name and its NCBI accession number (ideally the RefSeq number), and we will design the target
sequence using our proven algorithm and make the DNA constructs for you. We generally deliver the finished constructs in 15 business days and ship them to you as
glycerol stocks via expressed mail. If you order 3 or more constructs for each gene target, we offer a "Service Guarantee*"
If you already have the target sequence either from your siRNA or from published literature, we can simply clone them into one of our expression systems for you
under the "Construction Service" order.
Click on the catalog number link for detailed information of each type of services and place your order correspondingly.
The pRetro shRNA expression vectors are constructed from the MSCV retrovial vector whose backbone is derived from the Murine Embryonic Stem Cell Virus
(MSCV). The MSCV vector is a self-inactivating retroviral vector optimized for introducing and expressing target genes into pluripotent cell lines. When produced
from helper packaging cells such as Phoenix cells (commercially available from BD Clontech), MSCV vector-derived viruses can be used to infect murine or rat cells
(Ecotropic packaging line), or most other mammalian cells including that of human (Amphotropic packaging line). The MSCV vector carries a specially designed
long terminal repeat (LTR) from the murine stem cell PCMV virus, which differs from the MoMuLV LTR used in many other retroviral vectors by several point
mutations, and a deletion. As the result of these changes, the PCMV LTR drives high-level, constitutive expression of the target gene in stem cells or other
mammalian cell lines. Upon transfection into packaging cells, the pRetro vectors express, either transiently or stably, viral RNA transcripts to be packaged into
infectious but replication incompetent virus particles. The shRNA encoded in pRetro vectors can also be delivered either by standard transfection approach. A
compounded puromycin resistance-internal ribosome entry site-green fluorescence protein (PIG) cassette is inserted behind the phosphoglycerate kinase (PGK)
promoter in these vectors, allowing for antibiotic selection and green fluorescence labeling in infected cells.
Reference
1. Hawley RG, Lieu FH, Fong AZ, Hawley TS. (1994) Versatile retroviral vectors for potential use in gene therapy Gene Ther. 1:136-8.
2. Pear WS, Nolan GP, Scott ML, Baltimore D. (1993) Production of high-titer helper-free retroviruses by transient transfection. PNAS 90:8392-6.
The pLenti shRNA expression vector is derived from the third generation Lentiviral expression system. The preintegration complex of HIV-1 virus is recognized by
the cell nuclear import machinery and actively transported through the nucleopore due to karyophilic signals present on three viral proteins, MA Vpr and Integrase.
Thus, the HIV-1 derived Lenti virus can infect both replicating as well as non-relicating cells , making it an ideal vector for gene delivery. Some of the cell types
that are efficiently infected by lentivirus include neuronal cells or tissue explants, lymphocytes, keratinocytes, oesteoblasts and osteoclasts. We currently offer
pLenti-hU6BX, and pLenti-H1G for delivering shRNAs in vivo by lentivirus. The shRNA expression constructs generated in these vectors are fully compatible with the
Virapower packaging mix (K4970-00, InVitrogen). The Lentiviruses produced with this packaging system do not carry or express ANY viral genes and the lentivirus
itself cannot replicate because of built-in safety features. However, users should observe NIH and their own institutional guidelines for working with such viral
systems. The typical titer of the packaged Lentivirus is between 1 x 10^5 and 1 x 10^6 cfu/ml unconcentrated, or up to 1 x 10^9 cfu/ml after concentration. We
suggest titering the lentivirus on HT1080 (InVitrogen) cells.
Reference
1. Dull, T et al. (1998) A third-generation of Lentiviral vector with a conditional packaging system J. Virol. 72: 8463-8471.
2. Wang KC, Kim JA, Sivasankaran R, Segal R, He Z. (2002) P75 interacts with the Nogo receptor as a co-receptor for Nogo, MAG and OMgp. Nature
420(6911)74-78
