Analysis of gene expression, copy number and palindrome formation with a Dt40 enriched cDNA microarray

Abstract

DT40 presents a unique opportunity to exploit newly available tools for chicken genomic analysis. A 13K chicken cDNA microarray representing 11447 non-overlapping ESTs has been developed. This array detects expression of 7086 DT40 genes of which_644 are over-expressed 3-fold or greater and 1585 are under-expressed 3-fold or greater relative to normal post-hatch bursal cell populations. Changes in RNA expression due to single gene alterations can be detected by expression profiling. For example, by this method, over expression of the oncogenic micro RNA bic up-regulates expression of VBP, a known regulator of Avian Leukosis Virus LTR- driven transcription with very little additional expression change, A degree of cytogenetic abnormality and instability of DT40 cells has been observed, which is characterized at the fine structure level using microarray-based comparative genome hybridization (array-CGH). The relationship between gene copy number and RNA expression levels can be assessed in the same tissue samples using the same microarray. A newly introduced technique for genome-wide analysis of palindrome formation (GAPF) detects long inverted repeats, or palindromes, which are early events in gene amplification and possibly other DNA structural change. Since both array CGH-detected copy number changes and GAPF-detected palindromes are abundant in DT40, these techniques, coupled with targeted gene deletion and replacement, may provide a powerful tool for analysis of genomic instability and its underlying genetic mechanisms.