Genomic imprinting is a uniquely mammalian process by which two alleles of an autosomal gene are differentially expressed depending on their parent of origin. Imprinted genes are often found in clusters and it is thought that gene expression within the cluster is regulated by a shared mechanism. One imprinted gene cluster is the Dlk1-Gtl2 locus, located on mouse chromosome 12 (human chromosome 14q32). Proper expression of the genes at the imprinted Delta-like 1 (Dlk1) - Maternally expressed gene 3 (Meg3) locus is required for normal fetal development in mammals. The exact mechanisms that regulate genomic imprinting and control tissue-specific gene expression at the Dlk1-Meg3 locus are currently unknown, but are suspected to involve differential methylation of CpG dinucleotides (DMRs) which can suppress or activate gene expression. Three DMRs reside at this locus and acquire paternal-specific methylation that is involved in imprinting regulation. We have used a combination of mouse expression assays to localize a subset of the cis-regulatory elements required for Dlk1 expression in the mouse. Cross-species DNA sequence conservation was used to identify -regulatory elements contained within the sequence upstream of Dlk1 that can function as enhancers using a luciferase expression assay. Two of these elements could drive expression of a β-galactosidase reporter transgene in Dlk1 expressing tissues in the mouse; therefore, the sequence proximal to Dlk1 contains at least two discrete cis-regulatory elements that potentially regulate tissue specificity of Dlk1 expression. In an independent set of experiments imprinting control elements were analyzed. Two targeted deletions were designed to separately remove the mouse Gtl2 DMR and the mouse Gtl2 gene. Improper homologous recombination generated an allele termed ΔGtl2Dup with an insertion of the targeting vector upstream of Gtl2 that caused a duplication of the Gtl2 DMR sequence. Although not the intended mutation, the ΔGtl2Dup allele displayed embryonic lethality and loss of gene expression upon maternal transmission and was homozygous lethal by embryonic day 16. However, as a result of the Gtl2 DMR duplication, the results were not interpretable with regard to imprinting regulation and were not analyzed further.