Question 4
You've heard about this cool technique for targeted recombination into embryonic stem (ES) cells that allows you to create a null allele or a conditional allele more or less at the same time. As your friend explained it to you, you first carry out standard gene targeting into ES cells by homologous recombination, as shown below. The Neo gene, which codes for resistance to the antibiotic neomycin, allows selection for ES cells that have incorporated the vector. These cells can then be screened by Southern blotting for those that have undergone a targeted event. The really cool part is to flank the Neo gene and an adjacent exon or two with lox sites. This technique is commonly referred to as "floxing". Once the modified ES cells have been identified, they can be exposed to the Cre recombinase, which promotes site-specific recombination between pairs of lox sites. One advantage is that it allows you to get rid of the Neo gene and any bacterial DNA segments, which can sometimes influence the phenotype.
a) Draw all possible products you might get from expression of Cre in modified ES cells that carry three lox sites, as indicated above.
b) Which product(s) would be a null allele?
c) Which product(s) would have a pair or lox sites but be an otherwise normal allele (mark product(s) with an asterisk or star)?
c) If you had one mouse that expressed Cre under the control of a tissue specific promoter, can you use the allele in Part C as a conditional allele; that is, one whose defect is expressed only in a particular tissue?