Figure 2.

Overview of procedure for generating BAC15 and BAC16 reporter constructs. BACs were obtained from a BAC library maintained in the DH10B strain of E. coli (1). BACs were then purified and transformed into strain SW102 (or EL250; not shown) competent for recombineering (2). Primers with overhangs complementary to the targeted BAC region (homology arms H1 and H2) were used to amplify a cassette containing the GFP reporter and an ampicillin resistance gene (Amp) from plasmid pIS-GATA2-GFP using PCR. The insert amplified with primers F1 and R1 was used for BAC15 and did not contain a promoter, whereas the insert amplified with primers F2, R2 was used for BAC16 and contained the GATA2 minimal promoter (3).The insert was transformed into SW102 bacteria containing the appropriate BAC to allow for recombineering (4).