A modular lentiviral and retroviral construction system to rapidly generate vectors for gene expression and gene knockdown in vitro and in vivo.

Geiling B, Vandal G, Posner AR, de Bruyns A, Dutchak KL, Garnett S, Dankort D
PLoS One. 2013 Oct 11;8(10):e76279. doi: 10.1371/journal.pone.0076279. PubMed Article

Dankort Lab pLEG/pREG Modular Viral Vector System

The LEG ( Lentiviral Expression Gateway) is system a flexible vector system to allow the rapid production of lentiviral and retroviral (REG) vectors that express cDNAs with or without inhibitory RNAs (shRNAmirs) using Gateway technology.

This modular system consists of: a vector backbone (module 1), a cDNA contained in a standard pEntry vector (module 2), genetic markers or fluorescent proteins are encoded downstream of internal ribosome entry sequences and are flanked by attR2 and attL3 sites contained in a pBEG vector (module 3), and, an optional miR30-embedded shRNA flanked by attR3 and attL4 sites (module 4). These modules are combined using a multisite Gateway LR reaction with a destination vector. Destination vectors can be either a pLEX-based lentiviral vector (pLEG R1-R3 or pLEG R1-R4) or a pQCxix-based retroviral expression vector (pREG R1-R3) or pREG R1-R4). Each vector contains a self-inactivating 3'LTR.

The configuration allows the cDNA, genetic marker or fluorescent protein and miRNA to be cloned while maintaining precise order, spacing and orientation into either a lentiviral or retroviral destination vector. cDNAs may be maintained in pEntry vectors (AttL1 and AttL2) and the same plasmids used for multiple LR reactions to rapidly produce a large number of viral expression vectors with variations in selectable marker or miRNA expression (conversely the cDNA could be varied) without the need to characterize each plasmid. Novel miRNAs may be generated and triaged efficiently using a pCheck (AttR1 and AttR2) plasmid. To do this cDNA (from pEntry vectors) are cloned into pCheck R1-R2 behind Renilla luciferase by standard Gateway LR reaction and then targeted with a candidate shRNAmir.

Browse LEG system plasmids

Module 1	Destination vectors: pLEG second generation lentiviral vectors and pREG retroviral vectors for three or four plasmid recombination reactions
Module 2	pENTRY vector contains cDNA
Module 3	pBEG R2-L3 vectors contain an ires and selectable marker
Module 4	pBEG R3-miRNA-L4 contains miR-30 based shRNA for specific gene knockdown

Expressing cDNA with the marker of your choice (example of a three plasmid recombination). Choose a vector (here a lentiviral vector), provide a cDNA (in an Entry vector), and choose the desired marker in pBEG R2-L3 (here puromycin resistance).The resulting viral vector can be transduced into cells where cDNA expression is encoded on the same bi-cistronic mRNA with the selectable marker.

Figure 1: Viral vector is generated through a Gateway LR recombination reaction. Virus is produced by transient transfection using second generation packaging plasmids and cells are transduced.

Expressing cDNA and one or more shRNAs with the marker of your choice (example of a four plasmid recombination). Choose a vector (here a lentiviral vector), provide a cDNA (in an Entry vector), choose the desired marker in pBEG R2-L3 (here eGFP), and a shRNA cloned into pBEG R3-shRNAmir-L4.The resulting viral vector can be transduced into cells where cDNA expression and shRNA is contained within the same multifunctional mRNA along with the selectable marker.

Figure 2: Viral vector is generated through a Gateway LR recombination reaction. Virus is produced by transient transfection using second generation packaging plasmids and cells are transduced.

Testing shRNA efficacy using the dual luciferase reporter.

Figure 3: The target cDNA contained in a standard pEntry vector is cloned via Gateway LR reaction into pCheck2 Dest (R1-R2) downstream of Renilla luciferase.

Figure 4: Two different shRNAs are compared by normalizing for transfection efficiency via Firefly Luciferase and then for specific knockdown by assessing Renilla Luciferase levels.

Plasmids from Article

ID	Plasmid	Purpose
48942	pBEG R1-ChlorR-R3 #BGV148	Contains the Gateway selection cassette (ccdB ChlorR) flanked by AttR1 and AttR3 sites for the creation of Destination vectors
48943	pBEG R1-ChlorR-R4 #BGV049	Contains the Gateway selection cassette (ccdB ChlorR) flanked by AttR1 and AttR4 sites for the creation of Destination vectors
48952	pBEG R2-L3 #BGV193	For inserting selection gene between AttR2 and AttL3 sites for use in Gateway recombination cloning system (corresponds to a module 3 plasmid)
48953	pBEG R3-miRNA(ccdB)-L4 #BGV228	To create an AttR3-AttL4 flanked Entry vector encoding a miR30-embedded shRNA to knockdown targeted gene expression. (This corresponds to a module 4 plasmid).
48954	pBEG R3-L4 #BGV194	For inserting selection gene between AttR3 and AttL4 sites for use in Gateway recombination cloning system (corresponds to a module 3 plasmid)
48955	pCheck2 Dest (R1-R2) #BGV129	Destination dual luciferase reporter plasmid for checking effectiveness of shRNA. Compatible with standard AttL1-AttL2 Gateway Entry vectors.
48956	pLEG(R1-R3) #BGV149	Lentiviral Destination vector containing AttR1-AttR3 site. For use with three-way LR reaction with cDNA in entry vector and a marker in pBEG R2-L3 plasmids (module 2 and module 3 plasmids).
48957	pLEG(R1-R4) #BGV057	Lentiviral Destination vector containing AttR1-AttR4 site. Use with 4-way LR reaction with cDNA in entry vector, marker in pBEG R2-L3 plasmids and shRNA in pBEG R3-L4 plasmid (module 2,3,and 4)
48958	pREG(R1-R3) #BGV141	Retroviral Destination vector containing AttR1-AttR3 site. For use with three-way LR reaction with cDNA in entry vector and a marker in pBEG R2-L3 plasmids (module 2 and module 3 plasmids).
48973	pBEG R2-dsRed-L3 #BGV203	Supplies the selection marker (dsRed) and IRES with flanked by AttR2 and AttL3 sites for Gateway cloning recombination reaction. (This corresponds to a module 3 plasmid).
48977	pBEG R2-eCFP-L3 #BGV202	Supplies the selection marker (eCFP) and IRES with flanked by AttR2 and AttL3 sites for Gateway cloning recombination reaction. (This corresponds to a module 3 plasmid).
48979	pBEG R2-eGFP-L3 #BGV201	Supplies the selection marker (eGFP) and IRES with flanked by AttR2 and AttL3 sites for Gateway cloning recombination reaction. (This corresponds to a module 3 plasmid).
48982	pBEG R2-iBlast-L3 #BGV204	Supplies the selection marker (blasticidin resistance) and IRES with flanked by AttR2 and AttL3 sites for Gateway cloning recombination reaction. (This corresponds to a module 3 plasmid).
48985	pBEG R2-iHygro-L3 #BGV206	Supplies the selection marker (hygromycin resistance) and IRES with flanked by AttR2 and AttL3 sites for Gateway cloning recombination reaction. (This corresponds to a module 3 plasmid).
48986	pBEG R2-iNeo-L3 #BGV207	Supplies the selection marker (neomycin resistance) and IRES with flanked by AttR2 and AttL3 sites for Gateway cloning recombination reaction. (This corresponds to a module 3 plasmid).
48987	pBEG R2-iPuro-L3 #BGV205	Supplies the selection marker (puromycin resistance) and IRES with flanked by AttR2 and AttL3 sites for Gateway cloning recombination reaction. (This corresponds to a module 3 plasmid).
48991	pREG(R1-R4) #BGV059	Retroviral Destination vector containing AttR1-AttR4 site. Use with 4-way LR reaction with cDNA in entry vector, marker in pBEG R2-L3 plasmids and shRNA in pBEG R3-L4 plasmid (module 2,3,and 4)
48992	pBEG R2-iCreT2ALuc-L3 #BGV208	Supplies Cre recombinase and Luciferase and IRES with flanked by AttR2 and AttL3 sites for Gateway cloning recombination reaction. (This corresponds to a module 3 plasmid).