, 1988,

Mendez et al., 1997 and Nicholson et al., 1999). In transgenic animals, sizeable germline regions performed better in rearrangement and expression (Xian et al., 1998), nevertheless, rodents with fully human IgH transloci often failed to produce high affinity binders after multiple immunizations (Green and Jakobovits, 1998 and Pruzina et al., 2011). The suboptimal performance of a human IgH locus in transgenic mice, in respect of antibody yield and immune response, was attributed to the imperfect interaction of the human constant region of membrane Ig with the endogenous rodent cellular signaling machinery (Pruzina et al., 2011). This was supported by work in transgenic rats, LDK378 cost carrying human VH, D and JH gene segments linked to the rat C-region locus, which displayed IgG immune responses very similar to wild type controls (Osborn et al., 2013). In these transgenic animals a large part of the rat C-region was included in conjunction with ~ 30 kb downstream of Cα containing the

3′ enhancer regulatory region, termed 3′RR (Vincent-Fabert et al., 2010). It was reasoned that intergenic regions containing cis-acting control sequences might be important in their entirety to secure class-switch recombination and hypermutation. The size of the complete human IgH locus, ~ 1.3 Mb with 38–46 VH, ~ 23 D and 6 JH segments (Hofker et al., 1989 and Matsuda et al., 1998), provides an immense challenge for engineering of multi-gene constructs and their germline integration by DNA microinjection into fertilized eggs. This can be partly overcome by using a previous finding where PR 171 co-injection of multiple DNA constructs with homologous overlaps frequently led to co-integration into the genome (Bruggemann et al., 1991 and Wagner et al., 1996). The use of large restriction

fragments from modified bacterial CYTH4 artificial chromosomes (BACs) with terminal homology sequence enabled a functional Ig locus to be assembled (Osborn et al., 2013). Successful homologous or tandem integration could be verified by transcript analysis, which showed productive rearrangement of diverse VH-D-JH-Cγ products brought together from segments accommodated on several BACs. Here we compare chimeric human IgH expression from four transgenic rat-lines with identical human VH, D and JH segments but different rat C-region and 3′ enhancer sequence. We found that there is flexibility in the positioning of the C-genes but that the region downstream of Cα containing multiple transcriptional enhancer elements resulted in optimal immune response, class-switch recombination and somatic hypermutation. For the construction of the HC10 translocus, the rat genomic region from BAC clone CH230-408M5 (Invitrogen), including Cμ, Cδ and the region up to the γ2c switch region on a ~ 49 kb fragment, was extended with a 100 bp homology arm corresponding to the sequence immediately upstream of the rat γ2b switch region using the Red/ET recombination method (Gene Bridges GmbH, Heidelberg, Germany).