Floxed fragments, which contained the 800-bp simian virus 40 (splice/polyadenylylationlation signal [36] and a neomycin resistance cassette for positive selection, were inserted between the 5' and 3' fragments for homologous recombination and in the same orientation relative to the Pold1 gene.
Homology regions covering 6.6 kb upstream of Dmp1 exon 6 and 8.2 kb downstream of exon 6 were subcloned and FRT-flanked Neo resistance positive selection cassette was inserted into 179 bp downstream of exon 6.
In brief, a loxP-flanked neo selection cassette was inserted into the first exon of the Tbx3 locus by homologous recombination in J1 ES cells.
The loxP flanked neomycin resistance - thymidin kinase gene (neo-tk) selection cassette was inserted together with a 141 bp wt cDNA fragment of the β1 integrin gene coding for the entire cytoplasmic tail of the β1 integrin chain including the stop codon, into exon 15, just behind and in frame with the transmembrane coding region.
A PGK-neo selection cassette was inserted downstream of exon 1.
In this study, the IMR variable derived from our Heckman selection model was inserted into the initial PCS and MCS models.
First, an RPSL/neo selection cassette was inserted into the region of interest, and then replaced with DNA containing the appropriate sequence.
For our modular approach, a DESTination cassette containing attR1 recombination site, ccdB lethality selection gene, chloramphenical resistance gene, and attR2 recombination site, which allows for easier ligase-free att site recombination with removal of cassette selection genes, was inserted just downstream of the T7 promoter in the first multiple cloning site (MCS 1) in DUET vectors (Table 2).
For both replacement vectors the positive selection cassette, pgk-neo, was inserted into the unique BamHI site ∼180 bp distal to exon 7 in the antisense orientation to Smn transcription.
The positive selection cassette, flox-pgk-neo was inserted into the unique BamHI site 180 bp distal to exon 7.
