1. Retroviral Pseudotransduction for Targeted Cell Manipulation
Melanie Galla, Elke Will, Janine Kraunus, Lei Chen, Christopher Baum 
Molecular Cell 
Volume 16, Issue 2, Pages (October 2004)
DOI: /j.molcel

2. Figure 1
Retroviral Vector Mutants Used for Pseudotransduction Experiments, Expressing Either nlsCre or EGFP
(A) SF91 contains all cis elements required for RT and integration. Mutant aPBS contains a defective PBS, dPBS lacks the PBS required for RT, and dU5 lacks the att signal required for integration.
(B) Compared with SF91-EGFP, mutants aPBS, dPBS, and dU5 show a greatly reduced incidence of stable EGFP transduction. NIH3T3 cells were analyzed 5 days after exposure to particles.
(C) Pseudotransduction generates slightly increased EGFP expression between 5 and 13 hr after exposure of cells to retroviral particles, both with aPBS-EGFP and SF91-EGFP. Subsequently, EGFP expression (AU, arbitrary units) rises only when using SF91-EGFP, reflecting de novo synthesis of mRNA after provirus integration. In contrast, cells exposed to aPBS-EGFP return to baseline levels after ∼40 hr. SF91-Hyg-nlsCre was used as a non-EGFP expressing vector control. The inset shows histograms of mock-transduced cells (gray) and cells exposed to aPBS-EGFP (black line), 19 hr after treatment.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
Cre Expression Mediated by Pseudotransduction Is Dose Dependent and Avoids Toxic Side Effects
(A) Treatment of reporter cells with supernatants containing either the integrating vector SF91-nlsCre or the mutants aPBS-nlsCre, dPBS-nlsCre, or dU5-nlsCre resulted in highly efficient, dose-dependent recombination.
(B) Permanent expression of nlsCre by the integrating vector SF91-nlsCre leads to a competitive growth disadvantage of EGFP+ Cre+ cells during 20 days of culture in three independent experiments. No such effect is seen with mutant aPBS-nlsCre.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3 Counterselection of Cells Constitutively Expressing nlsCre
(A) Western blot of protein extracts harvested from target cells 9 days after particle exposure reveals persisting Cre expression from SF91-nlsCre (lane1). Prolonged exposure (3 hr instead of 1 min) revealed a low level of persisting Cre after use of mutant dU5-nlsCre (lane 2), but not with mutants dPBS-nlsCre (lane 3) and aPBS-nlsCre (lanes 4 and 5). EGFP expression from the converted Cre reporter allele is even stronger in cell populations treated with dPBS and aPBS. Lane 6 shows mock-transduced cells. The Ponceau stain (lower panel) shows equal protein load.
(B) Semiquantitative PCR to detect integrated nlsCre DNA. Genomic DNA from Sc-1 reporter cell populations was prepared 9 (a) or 22 days (b) after transduction and a PCR using nlsCre-specific primers was performed. Integrated nlsCre was detected in SF91-nlsCre (1)-, dU5-nlsCre (2)-, and dPBS-nlsCre (3)-treated cells. A much weaker signal was seen in aPBS-nlsCre-treated cells (4 and 5). This is consistent with the residual leakiness of these mutants (Figure 1B). A control PCR amplifying the EGFP sequence in the SFr-2 reporter allele indicated that equal amounts of genomic DNA were used (lower panel).
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
Pseudotransduction Is Receptor Mediated and Allows Targeting of Distinct Cells in a Mixed Population
Human HT1080 and murine Sc-1 cells containing the reporter allele SFr-2 were mixed and transduced using either ecotropic (middle panel) or RD114 pseudotyped particles (lower panel) containing either the integrating SF91-nlsCre or mutant aPBS-nlsCre vectors. Flow cytometry was performed 4 days after exposure to particles. The mixed cell population was stained with anti human HLA(A,B,C) antibody to identify the HT1080 subpopulation. The unstained Sc-1 cells are mostly located in the first channel detecting green fluorescence. EGFP expression induced by Cre activity is strictly dependent on the tropism of the envelope protein, and targeting is independent of the type of expression vector used (SF91-nlsCre, aPBS-nlsCre, or dPBS-nlsCre, data not shown). The different efficiencies reflect variations in vector preparations.
Molecular Cell  , DOI: ( /j.molcel )