**2.2 Genotyping**

20 Liver Tumors

predominant locus responsible for this difference to a 6 Mb region on Chromosome 17 (Peychal et al., 2009). This region corresponds to part of the chromosome 6p region

The C3H/HeJ strain (C3H), highly susceptible to both spontaneous and carcinogen-induced HCC, develops up to 50-fold more liver tumors than the B6 strain after a single carcinogen treatment (Drinkwater and Ginsler, 1986). We previously reported mapping the predominant locus responsible for this susceptibility, *Hcs7*, to distal Chromosome 1 (Bilger et al., 2004). This chromosomal region corresponds in part to the 1q21-24 chromosomal region amplified in up to 86% of human liver cancers (Lau and Guan, 2005; Chochi et al., 2009; Zhang et al., 2010). C3H alleles on mouse Chromosome 1 confer a dominant 15-fold increased susceptibility to male mice and a semi-dominant 5-fold increased susceptibility to

Here we analyze the effect of C3H Chromosome 1 alleles on spontaneous hepatocarcinogenesis, on apoptosis and mitosis after DEN treatment, and on preneoplastic lesion growth. We have mapped the *Hcs7* modifier to a 3.3 Mb region and used expression and

B6 and C3H mice were purchased from the Jackson Laboratory (Bar Harbor, Maine) and bred in our facilities. All mice were housed in plastic cages on corncob bedding (Bed O'Cobs, Anderson Cob Division, Maumee, OH), fed Purina 5020 diet (9% fat; St. Louis, MO), and given acidified tap water *ad libitum*. Mice were inspected daily and weighed monthly. Lines derived from the B6.C3H-Ch1 recombinant strain (Bilger et al., 2004) were generated as follows. B6 females were mated with B6.C3H-Ch1 males heterozygous for the congenic region. Markers between *D1Mit285* and *D1MIT17* were used to identify recombinants, and breakpoints were mapped more finely with additional markers. Males and females from a given recombinant line were then intercrossed to generate homozygotes. Heterozygous or homozygous mice were mated with B6 to generate heterozygous experimental progeny (Figures 1 and 3, and Table 3) or homozygotes were intercrossed to generate homozygous experimental progeny (Tables 1 and 2). Tested parental B6.C3H-Ch1 were homozygous.

Lines derived from B6.BR-Ch1 were generated by mating B6 females with B6.BR-Ch1 males heterozygous for the congenic region. The markers *D1Mit143*, *D1Mit17*, and two additional microsatellite markers at 172.9 and 176.9 Mb were used to identify recombinants. (The lack of SNPs or SSLPs between B6 and BR in this region prevented refinement of these breakpoints.) Heterozygotes were intercrossed to generate homozygous lines. B6 females were then crossed with homozygous congenic males to generate experimental progeny.

The C3H.B6-Ch1 line carrying B6 alleles for distal Chromosome 1 on a C3H genetic background was generated by crossing C3B6F1 (N1) mice with C3H mice and selecting C3H.B6 N2 mice carrying B6 alleles at *D1Mit143*, *D1Mit15*, *D1Mit166*, and *D1Mit461*. Backcrossing to C3H and selection of these B6 alleles was continued for eight additional generations to generate C3.B6(*D1Mit143-D1Mit461*)N10, or "C3H.B6-Ch1" (Figure 3) mice.

CGH arrays to identify the *Ifi202b* gene as a strong candidate for this locus.

amplified in the majority of late-stage HCC (Santos et al., 2007; Chochi et al., 2009).

female mice carrying C3H alleles (Bilger et al., 2004).

**2. Materials and methods** 

**2.1 Mice** 

DNA was prepared from spleen tissue by Proteinase K treatment/ammonium acetate/isopropyl alcohol precipitation as described (Bilger et al., 2004) or from spleen, tail, or toe tissue by alkaline lysis (modified from Truett et al., 2000). Approximately 2 mm3 of spleen or 10 mm3 of tail or toe tissue was incubated in 200 µl lysis solution (25 mM NaOH, 0.2 mM EDTA) at 95°C for 20-60 minutes and then neutralized by the addition of 200 µl 40 mM Tris (pH 8.0). After agitation for approximately 30 seconds and centrifugation for five minutes at 13.5 krpm, 0.25 to 2 µl of the supernatant was used directly for genotyping as described (Bilger et al., 2004).
