Conditional Reelin Knockout Mouse Generation

To create the targeting vector for the conditional Reelin knockout mouse line, pJB1 (described in (69 (link))) was used as background vector and murine SV129J ES cell DNA as template DNA to amplify the short homology arm (SA, 1.35 kb), the fragment containing the first exon (EX1, 1.25 kb), and the long homology arm (LA, 9 kb). To incorporate the first loxP site a ClaI and a PvuI restriction site were included in the SA reverse and the EX1 forward primer, respectively. By three-fragment ligation, the (1 (link)) SA (cut with SalI and ClaI) and (2 (link)) EX1 (cut with SalI and PvuI) fragments were combined by a (3 (link)) LoxP coding linker (two annealed oligos with sticky ends for ClaI and PvuI) and cloned between the Neo and HSVTK selection marker genes (XhoI site, compatible ends with SalI) of pJB1. The resulting vector was opened with NotI to insert the 9 kb LA fragment 3'-downstream of the loxP/FRT flanked Neo-cassette. The primers used were: SA_for, 5'- ATCGATGTCGACGGAAGTTTTGCTTCTTCCGGTG-3' (SalI); SA_rev, 5'-CAATCGATGTTGTTTGTCTACGCCGGCTGCAAC-3' (ClaI); EX1_for, 5'-CCTTCTCGCGATCGCGCGTCCTCGCAGAACGGGCAGCC-3' (PvuI); EX1_rev, 5'-GCGGCCGCGTCGACTGGGCAGCCACCGACCAAAGTGCTC -3' (SalI); LA_for, 5'-TGTTGCGGCCGCGGCGGCCAGTTAAAAGTTCCCGCTG-3' (NotI); LA_rev, 5'-GTCGACGCGGCCGCTTCCATAAAAGGGAAGAGCAAGATG-3' (NotI). The oligos used were: LoxP_for, 5'-CGATAACTTCGTATAGCATACATTATACGAAGTTATAT-3'; LoxP_rev, 5'-CGATATAACTTCGTATAATGTATGCTATACGAAGTTATCGAT-3'. The final construct containing the SA-LoxP-EX1-LoxP-Neo-LoxP-LA-HSVTK cassette was linearized and electroporated into SV129J ES cells. Gene-targeting-positive ES cells (PCR screen) were injected into C57Bl/6J blastocysts resulting in chimeric mice. The chimeras were crossed to C57Bl/6J mice, resulting in Reln^fl/wt mice, verified by PCR and Southern blot. Heterozygous animals were backcrossed to Meox-Cre on the BL6 background to yield germline mutant reeler mice. Heterozygous animals were also backcrossed to CAG-^CreERT2 mice to obtain homozygous Reln^fl/fl; CAG-Cre mice. These were again backcrossed to Tg2576 mice and offspring used in the experiments were brother/sister crossed CAG-Cre hemizygous and Tg2576 hemizygous mice. To ensure a minimal effect on the behavioral results we have found, all animals that were compared were brother/sister crosses that only differ by the absence or presence of Cre recombinase. Moreover, discovering a robust response like the one discussed here in a mixed background, as opposed to an inbred one, further strengthens the validity of the conclusions, rather than diminishing them. Most importantly perhaps, it further supports the applicability of the results to the even more genetically heterogeneous human population.