Lecture 22: binary systems Readings : Hartwell Reference D: Drosophila melanogaster :

1. Lecture 22: binary systems Readings: Hartwell Reference D: Drosophila melanogaster: Genetic Portrait of the fruit fly, esp. 818-826. Hawley and Walker. Ch. 5.2 Mosaic Analysis

2. Gene Replacement This figure is drawn incorrectly! There are problems with polarity.

3. Gong and Rong. 2003. Targeting multi-cellular organisms. Current Opinion in Genetics & Development. 13: 215-220.

4. in vivo donor generation Gong and Rong. 2003. Targeting multi-cellular organisms. Current Opinion in Genetics & Development. 13: 215-220.

5. Gong and Rong. 2003. Targeting multi-cellular organisms. Current Opinion in Genetics & Development. 13: 215-220.

6. Mosaic Analysis

7. Mitotic recombination (right) compared with normal recombination (left). Alberts Molecular Biology of the Cell. Third Edition. Fig. 21-73

8. Mosaic Analysis with X-rays

14. The sev protein product is required in the R7 cell while the bossprotein is required in the R8 cell.

15. Mosaic Analysis with FLP recombinase

16. Oogenesis in Drosophila The oogonium divides by mitosis. Incomplete cytokinesis creates 16 cells interconnected by cytoplasmic bridges. The central cell will become the oocyte and the others will become nurse cells. The oocyte is surrounded by somatic cells known as follicle cells. It is the assymmetric distribution of materials from the follicle cells that is ultimately responsible for polarity in the embryo.

17. Mosaic Analysis: germline clones

18. "Flip out" method

19. Effect of ectopic expression of Hedgehog on Drosophila wing development. (a) An interruption cassette construct can be used to express Hedgehog ectopically. This construct is introduced into Drosophila along with one containing an inducible promoter linked to the recombinase gene. Expression of the hedgehog gene (orange) is disrupted by the intervening yellow gene (blue). Brief exposure to heat shock induces expression of the recombinase enzyme, which catalyzes recombination at the FRT sites, leading to excision of the yellow gene and fusion of the constitutive promoter to the hedgehog gene. (b) In the developing wing of a normal fly, Hedgehog is expressed in the posterior (P) compartment (orange). Expression of Hedgehog in a localized sector in the anterior (A) compartment during development leads to mirror image duplication of anterior structures. This effect is similar to that resulting from ectopic expression of Sonic Hedgehog in the anterior region of the chick limb bud [See K. Basler and G. Struhl, 1994, Nature 368:208.] Legend from Molecular Cell Biology.

21. Targeting Deletions and Duplications Chromosomal deletions can be engineered by targeting two genes with a bacteriophage loxP sequence. The two loxP sites are inserted at the ends of the desired deletion (at genes A and D in the figure; the superscript 'm' denotes the resulting mutated versions of these genes). The loxP sites may be targeted to the same chromosome (cis configuration) or, as in the system used by Lindsay et al.1, on different chromosomes of a homologous pair (trans configuration). Recombination between the two loxP sites is catalysed by the Cre recombinase, which is transiently expressed within the embryonic stem cells. In the trans configuration a deletion and a reciprocal duplication are produced. In the cis configuration the deleted fragment is lost from the cells because it lacks a centromere (a specialized region that is needed for correct segregation of chromosomes into daughter cells at cell division).

24. Gene content and order in the region commonly deleted in del22q11 patients, compared with that of a region of mouse chromosome 16. Red lines indicate genes with the same order and orientation in the two species, green lines indicate genes that have changed position during evolution and blue lines indicate genes that have conserved their relative order but with inverted centromere–telomere orientation. CLTCL is not present in mouse. Arrowheads indicate the transcriptional orientation of Es2 and Ufd1l, the two anchor points of the Df1 targeted deletion. This scheme has been compiled on the basis of published data and our unpublished results. Not to scale.

25. Top, the wild-type Es2 chromosomal locus. 8 out of 10 exons are indicated by small rectangles. The targeting vector is shown below the wild-type allele. The chromosome engineering cassette Hprt3' contains, from left to right, a PGK promoter, the first 2 exons of a human Hprt minigene20, an intron with a loxP site, and a PolII–neo cassette7. Below this is the targeted allele (Es2m) after homologous recombination. On the right is the diagnostic restriction pattern of a recombined cell line DNA digested with EcoRI and probed with the 3' probe. RI: EcoRI restriction sites.

26. Wild-type, targeting vector and targeted allele (Ufd1lm) of the Ufd1l locus. Only 5 of the 11 exons are indicated. The chromosome engineering cassette Hprt5' (ref. 7) contains a PGK–puro cassette (in the opposite orientation to the Ufd1l gene), an Hprt minigene intron containing a loxP site and exons 3–9 of the human Hprt minigene. Exons 2 and 3 are the first two coding exons of the Ufd1l gene. On the right is the diagnostic restriction pattern of a recombined cell line DNA digested with EcoRV and probed with the 5' probe. RV: EcoRV restriction sites.

27. Scheme showing the products of Cre-induced trans recombination between the loxP sites contained in the two complementary cassettes. ES cells with recombined chromosomes (right) are selected for Hprt function using HAT. These cells are also resistant to puromycin and G418. AB2.2 ES cells are Hprt deficient.

28. Targeting Deletions and Duplications Chromosomal deletions can be engineered by targeting two genes with a bacteriophage loxP sequence. The two loxP sites are inserted at the ends of the desired deletion (at genes A and D in the figure; the superscript 'm' denotes the resulting mutated versions of these genes). The loxP sites may be targeted to the same chromosome (cis configuration) or, as in the system used by Lindsay et al.1, on different chromosomes of a homologous pair (trans configuration). Recombination between the two loxP sites is catalysed by the Cre recombinase, which is transiently expressed within the embryonic stem cells. In the trans configuration a deletion and a reciprocal duplication are produced. In the cis configuration the deleted fragment is lost from the cells because it lacks a centromere (a specialized region that is needed for correct segregation of chromosomes into daughter cells at cell division).