Abstract: Method of isolating exonic gene segments of a eukaryotic gene by preparing a loaded exon trapping vector, bringing the loaded exon trapping vector in an environment in which transcription of DNA to RNA and RNA processing can occur, isolating processed RNA which includes exonic gene segments of the eukaryotic gene and, optionally, preparing a DNA copy of the isolated processed RNA. The loaded exon trapping vector comprises a large-insert DNA vector containing a segmented donor gene construct and, inserted in or close to said segmented donor gene construct, a target DNA fragment of at least 10 kb of eukaryotic genomic DNA. Use of vectors containing large genomic inserts allows to isolate exonic gene segments present in the correct transcriptional orientation as a complete set, without need for reordering individually isolated exons and verifying their continuity from isolated cDNAs.