SUPPLEMENTARY FIGURE LEGENDS

Asimakopoulos and Varmus, “Cell-specific Transduction of Prdm1-Expressing Lineages Mediated by a Receptor for Avian Leukosis Virus Subgroup B”

Supplementary Figure 1. Construction of the Prdm1:TVB-mRFP transgene by recA-mediated BAC modification, according to the method developed by Heintz and colleagues (14). Schematic diagram of the modification process. The native configuration of the parental BAC is depicted on top. The translation initiation ATG of Prdm1 is flanked by two homology boxes of approximately 500 bp each. In the first step of modification, a shuttle vector bearing the modification cassette (TVB-mRFP-polyA) flanked by the upstream and downstream homology boxes is incorporated into the parental BAC by recombination through one of the homology boxes (co-integration). Continued expression of the recombinase recA from the co-integrate allows a second recombination step through one of the homology boxes, thus eliminating the vector sequences. The process can lead to generation of a modified BAC that bears the modification cassette at the desired position. Genomic distances in this graph are not drawn to scale.

Supplementary Figure 2. Panel A. BAC modification by homologous recombination in E.coli- Co-integration. Southern blot screening for co-integrate clones. BAC DNA was digested with XmnI and hybridized to a probe corresponding to the downstream homology box (see Supplementary Figure 1 for location of probe). Successful co-integration of the shuttle vector leads to double representation of each homology box (see Supplementary Figure 1). The probe therefore detects two bands following successful co-integration (open arrow). All other bands correspond to the native BAC configuration.

Panel B. BAC modification by homologous recombination in E.coli- Resolution. Southern blot screening for co-integrate resolution. Continued expression of recA in the co-integrate (open arrow) allows a second step of recombination that removes the shuttle vector sequences and either results in incorporation of the modification cassette at the desired position demonstrated by a bandshift compared to the native configuration (solid arrow) or restores the native configuration.

Panel C. Exclusion of gross rearrangements following modification of the Prdm1 BAC, RPCI23-217C20. SapI digest of the parental BAC (lane 1) and the modified BAC (lanes 2 and 3). Modification introduces a new SapI restriction site that results in a smaller SapI fragment in the modified BAC (open double arrow) in lieu of the native fragment of larger size in the parental BAC (solid double arrow). All other SapI fragments are identical between parental BAC and modified BAC. Lane 4 is a molecular weight marker.

Supplementary Figure 3. In situ detection of the TVB-mRFP receptor in thymus and gut tissue from Prdm1:TVB-mRFP transgenic animals. A polyclonal antibody against red fluorescent protein (Rockland) was used for in situ detection of the TVB-mRFP fusion receptor. Panel A. Thymus section from a Prdm1:TVB-mRFP transgenic animal. A few scattered mRFP+ cells are seen in both cortex (ThyC) and medulla (ThyM). Magnification, 10X; counterstain, hematoxylin. Panel B. Multiple cells display mRFP staining (brown, DAB) in the lamina propria of the small bowel villi. Magnification, 10X; counterstain, hematoxylin.

Supplementary Figure 4. Expression of TVB is required for transduction by an ALV-vector bearing a subgroup B envelope. T-cell depleted splenocytes from transgenic animals as well as non-transgenic controls were in vitro-stimulated in the presence of lipopolysaccharide (LPS), interleukins-2 and -6 and transduced with an RCAS(envB)-GFP vector. At 36 hours post-induction, some splenocytes from the transgenic animal (Panel A) express sufficient amounts of TVB-mRFP protein to allow detection by immunocytochemistry. Expression of the fusion receptor was undetectable in splenocytes from the control animal (Panel B). Post-transduction, only splenocytes derived from the transgenic animal (Panel C) but not the non-transgenic control (Panel D) express GFP. The low percentage of transduced cells in this assay (approximately 0.05% of total splenocytes in Panel C) may reflect the limited number of target cells expressing the receptor at transduction. This is in contrast to the higher rates of transduction observed in fractions enriched for TVB-mRFP receptor expression by prior flow-sorting (Figure 3).

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