pFB and pFB-Neo Retroviral Vectors
INSTRUCTION MANUAL
Catalog #217563 (pFB Retroviral Vector) and #217561 (pFB-Neo Retroviral Vector) Revision A
For In Vitro Use Only
217561-12
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the inability to use this product.
O RDERING I NFORMATION AND T ECHNICAL S ERVICES
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pFB and pFB-Neo Retroviral Vectors
C ONTENTS
Materials Provided (1)
Storage Conditions (1)
Introduction (2)
The pFB Vector (3)
The pFB-Neo Vector (4)
pFB-Luciferase Control Vector (5)
Replication-Defective Retroviral Gene Transfer Systems (6)
Biosafety Considerations (6)
Ligation of Vector and Insert (7)
Ligation Guidelines (7)
Ligation Protocol (8)
Recombination and Host Strains for Subcloning and Propagation (9)
Primers (9)
Preparation of Media and Reagents (10)
References (10)
Supplemental References (10)
MSDS Information (10)
pFB and pFB-Neo Retroviral Vectors M ATERIALS P ROVIDED
Material provided
Catalog #217561 Catalog #217563
pFB-Neo retroviral vector 10 µg —
pFB retroviral vector — 10 µg
pFB-Luciferase expression control vector 10 µg 10
µg
S TORAGE C ONDITIONS
All components:–20°C
N OTICE TO P URCHASER
Use of the translation enhancer of the pFB-Neo vector is covered by U.S. Patent No. 4,937,190 and is limited to use solely for research purposes. Any other use of the translation enhancer of the pFB-Neo vector requires a license from WARF.
B IOSAFETY
C ONSIDERATIONS
The host range of a retrovirus is determined not by the vector DNA but by the specific env gene used to construct the packaging cell line. Viruses produced from amphotropic or polytropic packaging lines are capable of infecting human cells. The National Institutes of Health has designated retroviral vectors, such as MMLV, as Biosafety Level 2. Appropriate caution should be used in the production a
nd use of recombinant retrovirus. For more information see Biosafety in Microbiological and Biomedical Laboratories at www.doczj/doc/dba401755b8102d276a20029bd64783e08127d58.html
/od/ors/ds/pubs/ bmbl/contents.htm.
Revision A ? Agilent Technologies, Inc. 2008.
I NTRODUCTION
The Stratagene pFB and pFB-Neo plasmid vectors for retroviral gene
delivery and expression are derived from the Moloney murine leukemia
virus (MMLV) and can be used to produce high titer viral stocks with
MMLV-based packaging cell lines.1,2 Both vectors contain the bacterial
origin of replication and ampicillin-resistance gene from pBR322, an
extended MMLV packaging signal (ψ+), and a multiple cloning site (MCS)
located between the MMLV 5′ and 3′ long terminal repeat sequences
(LTRs), see Figures 1 and 2.
The pFB vector is a minimal MMLV-based vector that can accommodate
larger inserts than pFB-Neo. The pFB vector does not contain any
exogenous sequence other than the sequence of the MCS.
The pFB-Neo vector differs from the pFB vector only by the presence of a
cassette consisting of the internal ribosome entry site (IRES) from the
encephalomyocarditis virus (EMCV) and the neomycin-resistance gene
(neo r). The open reading frame (ORF) for the neo r gene is positioned
downstream of the MCS and follows the IRES. A dicistronic message
encoding the gene of interest and the neo r gene is expressed from the viral
promoter within the 5′ LTR.
克氏锥虫
The pFB Vector
Feature
Nucleotide position 5′-long terminal repeat (LTR)
209–760 transcription initiation (clockwise)
616 splice donor
818–822 ψ+ extended viral packaging signal
760–2046 gag gene (truncated)
1236–1723 splice acceptor
1751–1753 5′ retro primer binding site
2008–2028 multiple cloning site
2057–2086 3′ pFB primer binding site
2121–2101 3′-long terminal repeat (LTR)
2163–2756 pBR322 origin of replication
3237–3904 ampicillin resistance (bla ) ORF 4055–4912
F IGURE 1 Circular map and features for the pFB retroviral vector. The complete nucleotide sequenc
e and list of restriction sites can be found at www.doczj/doc/dba401755b8102d276a20029bd64783e08127d58.html .
EcoR I Sal I Xho I Not I
BamH I pFB Multiple Cloning Site Region (sequence shown 2057?086)
The pFB-Neo Vector
Feature
她是我的朋友教学设计Nucleotide Position 5′-long terminal repeat (LTR) 209–760 transcription initiation (clockwise)
616 splice donor
818–822 ψ+ extended viral packaging signal
760–2046 gag gene (truncated)
1236–1723 splice acceptor
1751–1753 5′ retro primer binding site
2008–2028 multiple cloning site
2057–2086 3′ pFB primer binding site
2147–2127 internal ribosome entry site (IRES)
2093–2586 neomycin resistance ORF
2763–3557 3′-long terminal repeat (LTR)
3617–4210 pBR322 origin of replication
4691–5358 ampicillin resistance (bla ) ORF 5509–6366
F IGURE 2 Circular map and features for the pFB-Neo retroviral vector. The complete nucleotide sequence and list of restriction sites can be found at www.doczj/doc/dba401755b8102d276a20029bd64783e08127d58.html . EcoR I Sal I Xho I Not I
BamH I pFB-Neo Multiple Cloning Site Region (sequence shown 2057?086)
pFB-L UCIFERASE C ONTROL V ECTOR
The pFB-Luc plasmid vector (Figure 3), which contains the luciferase gene,
is included with the vectors as an expression control. A retroviral vector
expressing a reporter gene is useful for optimizing transfection efficiency,
confirming viral production, as well as ascertaining whether or not a target
cell line can be infected by a given viral stock.
Figure 3 Circular map for the pFB-Luc control vector. The complete nucleotide sequence and list of r
estriction sites can be found at www.doczj/doc/dba401755b8102d276a20029bd64783e08127d58.html .
R EPLICATION-D EFECTIVE R ETROVIRAL G ENE T RANSFER S YSTEMS
Replication-defective retroviral vectors contain all of the cis elements
required for transcription of a gene of interest and packaging of the
transcripts into infectious viral particles.2 The retroviral vectors typically
consist of an E. coli plasmid backbone containing a pair of viral LTRs
between which the gene of interest is inserted. In order to generate
infectious virus particles that carry the gene of interest, specialized
packaging cell lines have been generated that contain chromosomally
integrated expression cassettes for viral proteins Gag, Pol, and Env, all of
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