Role of Intron I in Expression of the Human Factor IX Gene

Abstract

The first intron (intron I) of the human factor IX gene, which has been previously suggested of having an expression-augmenting activity, was systematically studied for its potential enhancer activity. When tested with the chloramphenicol acetyltransferase expression vector with a minimal factor IX promoter, subregions of intron I showed only marginal enhancing activities (1.7-1.9-fold enhancement at the highest). Smaller subregions encompassing nucleotides 5660-6350 of the intron sequence even showed some weak negative regulatory activities (approximately 50% suppression at the highest), while a cytomegalovirus enhancer sequence, which was used as the positive control, had a 7-fold enhancement. A set of three factor IX minigene expression vectors with the same factor IX promoter were then constructed: p-416FIXc which contained the factor IX cDNA, p-416FIXm1 which contained the factor IX cDNA with a largely truncated intron I, and p-416FIXm2 which contained the factor IX cDNA with the intron I sequence further truncated. The p-416FIXm1 and p-416FIXm2 constructs showed 7-9-fold higher expression activities than p-416FIXc. The elevated factor IX antigen levels agreed well with the grossly elevated factor IX clotting activity and mRNA levels. These results indicate that the expression enhancing activity of intron I is not due to specific enhancer elements present in the intron subsequences, but is due to functional splicing sequences present in the precursor mRNAs produced from the minigene constructs containing intron I. By being efficiently assembled into spliceosome complexes, transcripts with splicing sequences may be better protected in the nucleus from random degradations than those without such sequences.

Persistent expression of canine factor IX in hemophilia B canines

“…The intronic sequence is argued to promote FIX expression by stabilizing mRNA. 22 1.2 × 10 13 particles of rAAV/cFIX.In and 6 × 10 12 particles of rAAV/cFIX were produced by transient cotransfection of 293 cells. 23 This transfection scheme produced no detectable adenovirus (data not shown).…”

Direct intramuscular injection with recombinant AAV vectors results in sustained expression in a dog model of hemophilia

“…The hF.IX cDNA contained a portion of the first intron of the factor IX gene, previously reported to increase mRNA stability, 15 and the first 50 nucleotides of 3′ untranslated region. The CMV promoter element has been previously shown to have sustained activity in muscle after intramuscular administration of an AAV ␤-galactosidase-expressing vector.…”

“…Briefly, the human factor IX cDNA (hF.IX) including a truncated portion of the first intron was derived from p416FIXm1 kindly provided by Dr K Kurachi (University of Michigan). 15 Digestion with BamHI yielded the hF.IXm1 minigene which contains the entire coding region of hF.IX cDNA and 981bp and 443 bp sequences from the 5′ and 3′-end regions of the factor IX first intron, respectively. The presence of intron I sequence has been reported to improve stability of F.IX mRNA.…”

“…The presence of intron I sequence has been reported to improve stability of F.IX mRNA. 15 The AAV vector plasmid pTR-UF2 35 was digested with BamHI to yield a vector backbone with 5′ and 3′ AAV inverted terminal repeat structures, with the CMV immediate-early promoter/enhancer upstream of the BamHI site, and the bovine growth hormone polyadenylation signal immediately downstream of the BamHI site. The FIXm1 minigene was subcloned into the BamHI site of the AAV vector backbone yielding pTRhFIXm1.…”

Mutant Bik expression mediated by the enhanced minimal topoisomerase IIα promoter selectively suppressed breast tumors in an animal model

“…The full-length mutant fragment, mICB1-T2A, was obtained in secondary PCR by using the mixture of the primary PCR products as template, and T2A-F and T2A-R as primers. The sequence of 214-645 bp in the pCDNA3.1 vector (Invitrogen), which contained the CMV enhancer sequence (À604/À173 in the CMV promoter), 27 was PCR amplified with primers 5 0 -TTGCTAGCTACGGGCCAGATATACGCGTT-3 0 and 5 0 -AACTCGAGTGAGTCAAACCGCTATCCACG-3 0 . The PCR product was digested with NheI/XhoI and subcloned at corresponding sites into topoIIa-pGL3, T2A182-pGL3 and T2A90-pGL3 to obtain CT572-pGL3, CT182-pGL3 and CT90-pGL3, respectively.…”