Immunoglobulin Genomics in the Guinea Pig Figure 1. The guinea pig IgH locus in scaffold 54. The guinea pig IgH locus length is approximately 4,302 kb from the most 59 VH segment (VH- 290p) to the most 39 end of a gene in genomic scaffold 54. Filled bars: potentially functional VH genes; open bars: VH pseudogenes and V-p; DH: diversity genes; JH: joining genes; m: IgM coding gene; d: IgD fragment; c: IgG coding gene; e: IgE coding gene and a: IgA coding gene. VH and DH genes are numbered based on the families and their order. The number before the dash (VHn) indicates the family, while the number after dash represents the genomic position. JH genes are numbered based on the order of their locations in the locus. Double slashes indicate gaps .10 kb. The unidirectional arrowheads above d gene segments in scaffold 54 indicate that its direction is opposite to the other heavy chain constant genes transcriptional direction. The scale does not apply the upper frame diagram. doi:10.1371/journal.pone.0039298.g001 genome.ucsc.edu/). FUZZNUC, an online software (http:// mobyle.pasteur.fr/cgi-bin/portal.py?#forms::fuzznuc), was used to find adjacent recombination signal sequences (RSSs) for the identification of variable, diversity, and joining gene segments. Five or more mismatched bases were allowed to cover all genes. Total Ribonucleic Acid (RNA) Extract and 39 RACE Total RNA was extracted from spleen tissues of three male HARTLY guinea pigs using TRIzol Reagent (Invitrogen, USA) and pooled equally. Animals were treated in accordance with the China Agricultural University on the protection of animals used for experimental and other scientific purposes. The study was approved by animal welfare committee of China Agricultural University with approval number XK257. Complementary deoxyribonucleic acid (cDNA) was synthesized from 1 mg of total RNA using 39RACE-reverse transcription primers. The first polymerase chain reaction (PCR) amplification of each Ig heavy chain constant region gene (m, c, e, and a) was performed using the corresponding sense primer and 39RACE- antisense primer 1. While the second PCR amplification was conducted using the corresponding sense primer and 39RACE- antisense primer 2. All primers are displayed in Table S1. Southern Blotting Analysis of Genomic DNA Sample Guinea pig liver genomic DNA was isolated according to the method as previously described [15]. The VH gene family-specific probes belonging to guinea pig VH1, VH2 and VH3 gene families were labeled using a PCR digoxygenin probe synthesis kit (Roche, Germany) with primers designed from VH1-95, VH2-17 and VH3- 157 (The primer sequences are displayed in Table S1). Ten micrograms of genomic DNA digested separately with BamH I, EcoR I, Hind III and Xba I (New England Biolabs, USA) were loaded into each well of a 0.8% agarose gel and electrophoresed for 12 h, transferred to a positively charged nylon membrane (Roche, Germany). Hybridization and detection were conducted following the manufacturer’s instructions. PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e39298