pRetro-Lib載體描述 pRetro-Lib, derived from Moloney murine leukemia virus (MoMuLV), is designed for delivery and expression of retroviral libraries or genes (1–3). Upon transfection into a packaging cell line, pRetro-Lib can transiently express, or integrate and stably express, a transcript containing the gene of interest. The 5' viral LTR in this vector contains a viral promoter that controls expression of genes cloned into the MCS. pRetro-Lib also contains the Col E1 origin of replication and E. coli Ampr gene for propagation and antibiotic selection in bacteria. pRetro-Lib can be used as an alternative vector in the construction of a cDNA library using the SMART? cDNA Library Construction Kit (Cat. No. 634901). Retroviral expression libraries can be constructed by directionally cloning the SfiI(dT)30 primed cDNA into the SfiIA/B sites of pRetro-Lib. Restriction digestion with SfiI will remove the Stuffer fragment, prior to cloning.
pRetro-Lib facilitates expression cloning in combination with the benefits of retroviral gene delivery. Upon transfection into a high-titer packaging cell line, RNA from the vector is packaged into infectious, replication-incompetent retroviral particles. That is, these retroviral particles can infect target cells and transmit the gene of interest (which is cloned between the viral LTR sequences), but cannot replicate within these cells since the cells lack the viral structural genes. pRetro-Lib does not contain the structural genes necessary for particle formation and replication, gag, pol, and env; these genes are stably integrated into the packaging line. Subsequent introduction of pRetro-Lib, containing Ψ+, transcription and processing elements, and the gene of interest produces high-titer, replication-incompetent infectious virus.
Propagation in E. coli:
Suitable host strains: DH5α, and other general purpose strains.
Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) to E. coli hosts.
E. coli replication origin: Col E1
Copy number: low
Plasmid incompatibility group: pBR322
NOTE: The viral supernatants produced by this retroviral vector could, depending on your cloned insert, contain
potentially hazardous recombinant virus. Due caution must be exercised in the production and handling of
recombinant retrovirus. Appropriate NIH, regional, and institutional guidelines apply. References: 1. Coffin, J. M. & Varmus, H. E., Eds. (1996) Retroviruses (Cold Spring Harbor Laboratory Press, NY).
2. Ausubel, F. M., et al. (1994) Current Protocols in Molecular Biology (Greene Publishing Associates, Inc. & John Wiley & Sons, Inc.).
3. Miller, A. D. & Rosman, G. J. (1989) BioTechniques 7:980–990.
