Class switch recombination was dramatically reduced for α and all four γ genes in transgenes lacking either the insulator region and HS4, or the insulator region, HS4, HS3B, and HS1,2. For most H genes, CSR was reduced to 1–5% of the levels of CSR from wild type transgenes (, , , ). For example, transgenic VDJCγ1 expression in lines #346 and #220 was indistinguishable from the background level of nontransgenic B cells (). Average secretion levels of 101 or 131 µg/ml of transgenic IgG1a from truncated transgenes compared to 12 µg/ml background levels in nontransgenic mice might suggest some small amount of CSR (). However, in both the assay for secreted IgG1 and the assay for expression of VDJCγ1 mRNA, the difference in transgenic γ1 expression from truncated H chain transgenes was not statistically different from background levels from nontransgenic B cells. With enough replication of careful experiments, one could probably determine if there were some level of CSR remaining from the truncated H chain transgenes. We focused on the fact that CSR is severely disabled in both line #346 and line #220 transgenes, to less than 2% of wild type levels, and did not attempt to exactly quantify the magnitude of the impairment.
Expression of the transgenic VDJ sequences with endogenous Cγ or Cα sequences (Fig. 6ABC) complicated the quantification of the amount of CSR by the truncated transgenes. Mechanistic aspects of these putative “trans-recombination” events are of interest. Durdik, Guisti, and colleagues were able to detect products of trans-recombination after repeated immunization in vivo 
, but the rate of trans-recombination in light of strong selection by antigen was difficult to estimate. We would suggest that the rate of trans-recombination to the endogenous γ2a gene is at most
1% of the rate of CSR by a wild type transgene, because the efficiency of VDJCγ2a expression from the truncated transgenes is about 1% of that from the wild type transgene (), and trans-recombination can be about equal to CSR by the truncated transgene (, line #346). The mechanism of the trans-recombination is also an interesting subject for future studies. Even though some recombination events take place in switch regions 
, others can take place even if donor transgenes lack switch regions 
. Even though the majority of the recombination events require AID, a portion of the trans-recombination events may be independent of AID 
. It seems likely that the final products of trans-recombination arise from multiple recombination pathways 
The truncation of the H chain transgenes had a more variable effect on germline transcription. Germline transcription of some H chain genes was not altered (γ2b, ), whereas germline transcription of other H chain genes was greatly reduced (γ3 and α, ). Germline transcription of the γ1 and γ2a genes in line #346, with the larger truncation, was not substantially different from germline transcription of these two γ genes in intact H chain transgenes (). On the other hand, germline transcription of the γ1 and γ2a genes in line #220, with the truncation of HS4 and sequences more 3′, was dramatically reduced. One interpretation of these results would emphasize more the differences between line #346 and line #220 in the truncation of H chain transgenic sequences. Sequences between HS3A and HS4 may inhibit germline transcription of the γ1 and γ2a genes in the line #220 trangene, but since these sequences are deleted in line #346, germline transcription of these two H chain genes would precede at higher levels (). An alternative interpretation would emphasize the difference in chromosomal insertion site for these two H chain transgenes. Insertion site effects in line #346, resulting from positive control elements in sequences that flank the transgene, would increase the germline transcription of the γ1 and γ2a genes. A different version of this alternative interpretation is that negative control elements in the sequences that flank the transgene in line #220 inhibit expression of γ1 and γ2a germline transcripts.
Nevertheless, the functional differences between the transgenes in line #346 and in line #220 are minor compared to the deficiency they share–the nearly complete lack of CSR. The lack of CSR may result from negative elements that inhibit CSR in the sequences flanking the insertion site in both transgenic lines. Supporting the notion of an insertion site effect, the region deleted by both truncations includes elements with the structure and activities of an insulator element 
. Loss of this insulator region may make CSR activities sensitive to the transgene insertion site. It is important to note that the transgenic µ gene is expressed well in lines #346 and #220 ( and ). Hence, any potential insertion site effects do not alter transgene expression in general, but rather CSR and some aspects of germline transcription. A second interpretation for the lack of CSR in both transgenic lines is deletion of enhancers for CSR. In this regard, since the phenotypes of transgene germline transcription and CSR are more similar for lines #346 and #220 than they are different, it may be useful to consider that both HS4 and HS5-7 are deleted by the two truncations. In the endogenous locus, there is no evidence that either HS4 
or HS5-7 
include an enhancer element that is required for CSR or germline transcription. Experiments investigating enhancer activity 3′ of HS4 also argue against enhancer elements in this region that are strictly required for CSR 
. Therefore, if deletion of enhancer elements is the cause of the poor CSR in lines #346 and #220, one might surmise that at least two redundant elements, one in HS4 and a second in HS5-7 or in the sequences more 3′ were deleted as a result of the truncation. The hypothesis that two redundant regulatory elements are deleted in the truncation H chain transgenes has precedent in other studies of regulatory elements in the 3′ regulatory region. Deletion of a single enhancer element does not alter CSR 
, but deletion of two enhancers often reduces germline transcription and CSR of some H chain genes