Absence of internal ribosome entry site-mediated tissue specificity in the translation of a bicistronic transgene

Abstract

The 5' noncoding regions of the genomes of picornaviruses form a complex structure that directs cap-independent initiation of translation. This structure has been termed the internal ribosome entry site (IRES). The efficiency of translation initiation was shown, in vitro, to be influenced by the binding of cellular factors to the IRES. Hence, we hypothesized that the IRES might control picornavirus tropism. In order to test this possibility, we made a bicistronic construct in which translation of the luciferase gene is controlled by the IRES of Theiler's murine encephalomyelitis virus. In vitro, we observed that the IRES functions in various cell types and in macrophages, irrespective of their activation state. In vivo, we observed that the IRES is functional in different tissues of transgenic mice. Thus, it seems that the IRES is not an essential determinant of Theiler's virus tropism. On the other hand, the age of the mouse could be critical for IRES function. Indeed, the IRES was found to be more efficient in young mice. Picornavirus IRESs are becoming popular tools in transgenesis technology, since they allow the expression of two genes from the same transcription unit. Our results show that the Theiler's virus IRES is functional in cells of different origins and that it is thus a broad-spectrum tool. The possible age dependency of the IRES function, however, could be a drawback for gene expression in adult mice.

FIG. 1

(A) Schematic representation (not to scale) of the pCJ9 and pCJ12 vectors. For construction of the pCJ9 vector, the BamHI-SalI fragment carrying the CAT-IRES-L-LUCIFERASE construct was inserted between the corresponding sites of the pHMG plasmid (28). The cat gene was amplified by PCR with primers that introduced BamHI and EcoRV sites at its 5′ and 3′ extremities, respectively. The luc gene was also amplified by PCR. An HpaI restriction site was introduced four codons downstream from its 5′ extremity, and a SalI site was introduced at its 3′ extremity. For the IRES-L fragment, the IRES from plasmid pTMDA (26) was digested at its 5′ extremity by SmaI at the restriction site at position 12. An HpaI restriction site was inserted by site-directed mutagenesis in the L protein, four codons downstream from the initiator AUG. The IRES-L fragment was fused at its 5′ extremity to the cat gene by the EcoRV/SmaI junction and at its 3′ extremity to the luc gene by the HpaI/HpaI junction. For the pCJ12 vector, the IRES was deleted from the beginning to the Asp718 restriction site at position 930 in pTMDA. The cat gene was thus fused to the deleted IRES by the EcoRV/Asp718 junction, after filling in of the latter site with the Klenow enzyme. Restriction enzyme sites: EV, EcoRV; Sm, SmaI; Ba, BamHI; Hp, HpaI; Sa, SalI; As, Asp718. The minor splice donor sites are represented by the small triangles. SV40, simian virus 40. (B) The pCJ9 and pCJ12 vectors were transfected into the BHK-21 cell line, and expression of the Luc and CAT proteins was measured (in arbitrary units). Luc expression was normalized for equivalent amounts of CAT.

FIG. 2

Detection of Theiler’s virus in the tissues of FVB/N mice inoculated by different routes. The presence of the virus was detected by comparative RT-PCR followed by Southern blotting in 11 different tissues of nine mice (one mouse for each time point and inoculation route). Lanes: 1, heart; 2, lungs; 3, liver; 4, spleen; 5, esophagus; 6, mesenteries; 7, kidneys; 8, muscle; 9, brain; 10, spinal cord; 11, intestine. Data for four tissues of 18 additional mice are presented in Table 2. (Note that mice 12, 14, and 17 correspond to mice 3, 5, and 8 for the intraperitoneal route in Table 2, and mice 21, 22, and 26 correspond to mice 3, 4, and 8 for the intracerebral route in Table 2.)

FIG. 3

(A) Detection of the P_HMGCR-CAT-IRES-LUCIFERASE-_(A)n transgene in Therese mice by Southern blotting. DNA was digested with the PvuII restriction enzyme and analyzed by Southern blotting. Lanes: 1, transgenic DNA; 2, nontransgenic DNA plus 100 copies of the pCJ9 plasmid; 3, nontransgenic DNA. Digestion of transgenic DNA with PvuII generated the expected fragments of 4,262 and 1,248 bp and an additional fragment of 3,499 bp due to the insertion of transgenes in tandem. (B) Schematic representation (not to scale) of the P_HMGCR-CAT-IRES-LUCIFERASE-_(A)n DNA fragment after PvuII digestion.

FIG. 4

Northern blot of the CAT-IRES-Luc and β-actin transcripts from various transgenic tissues of Therese mice. (A) The CAT-IRES-Luc transcript was detected with a Luc probe. (B) The same membrane was hybridized with a β-actin probe. Molecular sizes (in kilobases) are indicated by arrows. Lanes: 1, stomach; 2, liver; 3, brain; 4, brain (nontransgenic mouse); 5, testis.

FIG. 5

Luc activities and levels of bicistronic mRNA in various tissues of transgenic mice. Luc activity was measured by a luminometric assay, and mRNA levels were evaluated by comparative RT-PCR amplifications. Results are shown for Therese mice that were 1 day (A), 1 week (B), 3 weeks (C), and more than 3 months (D) old. The data presented here should be considered per mouse and not compared between different mice, as explained in Materials and Methods. H, heart; Lu, lungs; Lv, liver; Sp, spleen; St, stomach; K, kidneys; Li, large intestine; B, brain; S, spinal cord; I, small intestine.

FIG. 6

Luc activity and bicistronic mRNA expression in Lucy mice. Luc activity and bicistronic mRNA expression in different tissues of a 1-week-old Lucy mouse (A) and an adult Lucy mouse (B) were compared. The comparative RT-PCR amplifications were hybridized by Southern blotting and quantified with a PhosphorImager. The quantity of bicistronic mRNA was normalized to the amount of β-actin mRNA. Scales are in arbitrary units. H, heart; Lu, lungs; Lv, liver; K, kidneys; B, brain.

FIG. 7

Luc activity and bicistronic mRNA expression in tissues of mice of different ages. For the evaluation of bicistronic mRNA levels, comparative RT-PCR amplifications were hybridized by Southern blotting and quantified with a PhosphorImager. The quantity of bicistronic mRNA was normalized to the amount of β-actin RNA. (A) Heart, Lucy mice; (B) brain, Lucy mice; (C) kidneys, Therese mice; (D) brain, Therese mice. Scales are in arbitrary units.