A new approach for studying gene regulation by distant DNA elements in transgenic mice - Expression of bacterial artificial chromosomes modified by RARE cleavage reveals that apolipoprotein B gene expression in the intestineis controlled by DNA sequences located more than 50 kb 5 ' to the structural gene

Apolipoprotein B (apo-B) plays a crucial role in the assembly of lipoprotei ns in the liver and the intestine. Here, we review how transgenic mouse exp ression studies with large genomic clones have been used to define distant cis-acting regulatory DNA sequences that control the expression of the apo- B gene. In early studies, apo-B transgenic mice were generated with similar to 80-kb P1 bacteriophage clones spanning either the human or the mouse ap o-B genes. Both the human and mouse clones directed high levels of transgen e expression in the liver, but transgene expression was absent in the intes tine. The absence of transgene expression in the intestine was surprising b ecause both P1 clones contained more than 11 kb of flanking sequences both 5' and 3' to the gene. Subsequently, we isolated and characterized 145-kb a nd 207-kb bacterial artificial chromosome (BAC) clones that spanned the hum an apo-B gene. Each of these BAC's contained extensive 5 and 3' flanking se quences and each directed spatially and physiologically appropriate apo-B g ene expression in the intestines of transgenic mice. To define the location of the sequences that control intestinal expression of the apo-B gene, we generated transgenic mice by co-microinjecting the similar to 80-kb P1 bact eriophage clone (which did not confer intestinal expression of apo-B) with either the 5' sequences or the 3' sequences from the 145-kb BAG. Analysis o f the apo-B expression pattern in those mice revealed that the DNA sequence s controlling intestinal expression were located 5' to the apo-B gene. Next , we used I ecd-assisted restriction endonuclease (RARE) cleavage to trunca te specific segments of the 5' and 3' flanking sequences from the 145-kb BA G. A series of the truncated BAC's containing different lengths of 5' and 3 ' sequences was used to generate more than 40 additional lines of human apo -B transgenic mice. Analysis of human apo-B gene expression in those mice d emonstrated that the sequences controlling the expression of the apo-B gene in the intestine are located more than 50 kb 5' to the apo-B gene. Our stu dies demonstrate that the RARE cleavage/transgenic expression strategy is a powerful approach for examining gene regulation by distant gene-regulatory elements.