BAC Modification through Serial or Simultaneous Use of CRE/Lox Technology
Figure 4
Sequential modification of a mouse Hoxb BAC MMP5 by serial recombination. (a) The top illustrates the organization of the MMP5 BAC containing a HoxB genomic fragment and the respective adjacent Hox genes targeted with four different fluorochrome reporter insertions through four rounds of serial modification. The middle row shows a single whole 9.5 dpc embryo examined under bright field (far right) and fluorescent illumination. This demonstrates that the BAC manipulations generated the four expected fluorescent proteins with appropriate activities in transgenic embryos. At the bottom, flat-mount preparations of the hindbrains dissected from this embryo show activity of the reporters in rhombomeric segments correlates with those expected for the endogenous genes. Hindbrain tissue imaged in the bottom row was taken from the region indicated by the white box in the bright field image in the row above on the far right. r4–7 = rhombomeres 4 to 7 and OV = otic vesicle. (b) It illustrates constructs and strategies for BAC targeting using fragments generated by PCR in combination with different fluorochrome containing selection cassettes (top). The targeting of the Hoxb4 locus with H2B-EGFP is given as an example. Homology-mediated targeting events are initiated based on primers containing homology to locations indicated in gray and black within the BAC which results in fluorochrome-selection cassette integration at specific locations. (c) At left (lane 1) electrophoresis of 1 kb Plus DNA ladder. Lanes 2–4 are NheI digestion of MMP5 BAC variants to monitor targeting events and BAC integrity by banding patterns. One kb Plus band sizes indicated on left. Lane 2 is the BAC MMP5 with an H2B-m Cherry insertion into Hoxb1. Lane 3 shows the changes in banding pattern when H2B-Venus is inserted into Hoxb2 as evidenced by the loss of a 7.9-kb fragment (red arrow) and the appearance of a 6.7- and 4.4-kb bands (green arrows). After Cre-mediated recombination to remove the selection cassette, a 9.4-kb band is now present and the 6.7- and 4.4-kb bands are now lost as shown in lane 4 as expected from proper targeting. The remaining bands in lanes 2–4 were identical indicating no additional rearrangements. Lost bands are indicated by red arrowheads and newly acquired bands by green arrowheads on the right of each image. (−), (+) numbers listed below each digest indicate the sizes of lost (−) and gained (+) bands in each BAC modification.
