Transgene analysis of switch recombination and murine gamma-1 germline transcription.

Abstract

B cells can express one of eight constant regions ($\mu, \delta, \gamma 3, \gamma 1, \gamma 2$b, $\gamma 2$a, $\varepsilon$, or $\alpha$) with the variable region on the immunoglobulin heavy chain they produce. Naive B cells express the $\mu$ constant region exons making IgM. In response to antigen a B cell can switch the heavy chain constant region it expresses from $\mu$ to any of the downstream isotypes except $\delta$. The B cell does this by a DNA recombination event called the immunoglobulin heavy chain switch. Prior to DNA recombination the genes involved are transcribed in the germline configuration in a process called germline transcription. This is believed to be a necessary event for switch recombination. Using a transgene system, we have isolated cis acting elements in the murine $\gamma 1$ gene which are important for $\gamma 1$ germline transcription. We have shown that a 4.4 kb fragment of the $\gamma 1$ gene is sufficient to mediate transcriptional regulation like the endogenous $\gamma 1$ gene. We have also constructed a second transgene which is 900 bp smaller than the first construct and lacks protein binding sites which might be important for transcriptional regulation. Analysis of a separate transgene construct, lacking the $\gamma 1$ switch region (S$\gamma 1$), suggests that S$\gamma 1$ is dispensable for transcriptional regulation but may function to increase the level of stable transcripts. In a separate line of experimentation we have attempted to isolate cis acting elements sufficient to mediate $\mu$ to $\gamma 1$ switch recombination. In order to do this, we designed a large transgene construct which encodes $\mu,\ \delta$ and $\gamma 1$ heavy chains. This construct can mediate $\mu$/$\gamma 1$ switch recombination and lead to the production of a $\gamma 1$ heavy chain. This event is dependent upon IL-4 and involves recombination of the switch regions. However, the efficiency of expression of IgG1, in terms of secreted antibody, from the transgene is lower than from the endogenous locus in wild type B cells. This suggests that the transgene is missing an element which is necessary to mediate efficient $\mu / \gamma 1$ switch recombination.