Supplemental methods

We used the pND1 vector to construct the targeting vector, which has neomycin (NEO) and diphtheria toxin (DT) cassettes for positive and negative selection, respectively. 1 This vector has a loxP site and two flippase recognition target (FRT) sites flanking the NEO cassette for potential removal by FLPe recombinase. Factor H gene fragments were amplified from 129/Sv mouse genomic DNA by using the Expand Long Template PCR system (Roche, Indianapolis, IN). The long arm was made from a 7kb fragment of the intron between SCR18 and SCR19 by PCR amplification and ligation into the PCR 2.1 vector (Invitrogen) using the following primers: 5’-GCGGCCGCCTGTGCAAGATGACAAATGTGGA-3’ and 5’CTCGAGCATTCCATGAAGAGATGAGAC-3’. This fragment was then digested with NotI and XhoI and subcloned into the pND1 vector upstream of the NEO cassette. The short arm sequence was 4.7kb in length and contained the 3’ end of the intron preceding SCR 19 through the middle of the exon containing SCR20. This sequence was PCR amplified and cloned into the PCR 2.1 vector using the following primers: 5’-GGTACCCTCTTTAGCCTTCTGTAGCA-3’ and 5’-GGTACCTCCACCATGATGATAATGGGC-3’. This sequence was then mutated to introduce two early stop codons (TCA-ACA to TAA-TGA) at the beginning of SCR 19 using the Stratagene QuickChange Site-Directed Mutagenesis kit (Agilent Technologies, CA). Once sequence was confirmed, the short arm fragment was digested using KpnI and subcloned into the pND1 vector downstream of the NEO cassette at the same restriction site.