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ÓSCAR ESCRIBANO Y COLS. AN. R. ACAD. NAC. FARM.

2. MATERIALS AND METHODS

2.1. Animals

IR(loxP/loxP) C57Bl/6 mice were created by homologous recombi-
nation using an IR gene targeting vector with lox P sites flanking exon
4 as previously described (11). Transgenic mice expressing a Cre
recombinase transgene under the control of the Mx1 promoter/
enhancer were purchased from Jackson Laboratory (Bar Harbour,
Maine, USA). The IR(loxP/loxP) mice were crossed with Mx-Cre mice to
obtain inducible LIRKO (iLIRKO) mice. After the suckling period,
iLIRKO mice were injected intraperitoneally with poly-Inositic-poly-
Cytidilic acid (500 mg/injection) to induce the interferon alpha
response and the consequent Mx1 promoter activation as previously
described (12). All animal experimentation described in this
manuscript was conducted according with accepted standards of
human animal care published by the National Institutes of Health.

2.2. Genotyping of the IR(loxP/loxP) transgenic mice

Genotyping of the mice was performed by PCR. Tail DNA (100-
200 ng) was amplified 30 cycles (40 seconds, 94° C; 40 seconds,
60° C; and 1 minute, 75° C) on a thermal cycler. Two primers
flanking the loxP site behind exon 4 of the IR were used: the forward
primer (5’-GATGTGCACCCCATGTCTG-3’) and the reverse primer (5’-
CTGAATAGCTGAGACCACAG-3’). A 320-bp band was obtained for
the floxed allele or a 280-bp band for the wild-type allele.

2.3. Genotyping of the Mx1-Cre transgenic mice

Mice were genotyped by PCR. Tail DNA (100-200 ng) was ampli-
fied 35 cycles (1 minute, 94° C; 1 minute, 60° C; and 1 minute, 72° C)
on a thermal cycler. To amplify the Mx1-Cre transgene (PCR product,
269 bp), primers SF-4 (5’-GCATAACCAGTGAAACAGCATTGCTG-3’)
and 69R (5’-GGACATGTTCAGGGATCGCCAGGCG-3’) were used.

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