Nemeth and Lowrey: A chromatin opening element increases !-globin expression
Figure 3. Analysis of integrated !-globin vector DNA. (a) Top: Vector schematic displaying Xho I (X) and Sal I (S) restriction sites and the range of sizes of the digestion products. Bottom: Integrity of retroviral vectors. Southern blot displaying intact !globin sequences for 5 out of 6 vectors. Human bone marrow DNA (H!) was digested with Pst I/Bgl II as a positive control. Genomic DNA from each pool was isolated and digested with Xho I/Sal I. (b) Multiple integration sites of retroviral pools. Genomic DNA isolated from r!G and rH!G pools was digested with Eco RI which cuts once within the vector.
To quantify the proportion of !-globin promoters accessible to restriction endonuclease digestion, and therefore in an open chromatin configuration, we performed restriction endonuclease assays on all pools generated from selected constructs. Intact nuclei were
performed restriction endonuclease assays on all pools generated from selected constructs. Intact nuclei were digested with Bln I, which uniquely digests at a single site within the !-globin promoter (Figure 5a).
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