The lac operator-repressor system is functional in the mouse

Abstract

We report the successful transfer of a fully functional lac operator-repressor gene regulatory system to the mouse. The key component is a lac repressor transgene that resembles a typical mammalian gene both in codon usage and structure and expresses functional levels of repressor protein in the animal. We used the repressor to regulate the expression of a mammalian reporter gene consisting of the tyrosinase promoter embedded with three short lac operator sequences and the tyrosinase coding sequence. Pigmentation of the mouse was controlled by the interaction of the lac repressor with the regulatable Tyrosinase transgene in a manner that was fully reversible by the lactose analog IPTG. Direct control of mammalian promoters by the lac repressor provides tight, reversible regulation, predictable levels of de-repressed expression, and the promise of reversible control of the endogenous genome.

Figure 1

Schematic representation of lacI transgenes (A) and target transgenes (B). (A) The lacI transgenes. All constructs are driven by a 4.3-kb fragment of the human β-actin promoter (shown in white; only the 3′ region of the promoter is represented), containing an intron indicated by the splice donor (d) and acceptor (a) sites. For each construct, the coding region for the lac repressor is derived from segments of the wtlacI (W, shown in yellow) and synlacI (S, shown in blue) coding regions as indicated. The first four chimeras were made by exchanging the linker between the promoter and coding region, and/or the 5′ part of the coding region (5′C1–4) between W and S. The next four chimeras were made by exchanging 3′ segments of the coding region (3′C1–4). In the last two constructs, M (W coding region) and R (3′C4 coding region), the coding region is flanked by segments of the rabbit β-globin locus (shown in red). (alt. a, potential alternative splice acceptor sites present in the lacI coding region; E and P, EcoRV and PvuII restriction enzyme sites; arrows indicate position of primers used for the PCR shown in Fig. 2). (B) The lac operator containing target transgenes. (1) SVOZ. The SV40 early promoter containing a single, symmetric lac operator drives expression of the β-galactosidase (lacZ) reporter gene, which contains the endogenous O_z operator. (2) The regulatable Tyrosinase transgene (Tyr^lacO). Three lac operators have been introduced into the murine tyrosinase promoter. The primary operator was centered just downstream of the start of transcription by changing the endogenous promoter sequence; two additional operators were inserted 176 bp and 526 bp upstream. The modified promoter drives expression of the wt murine tyrosinase cDNA.

Figure 2

Modifications to the coding region alter splicing and function of the lac repressor transgenes. (A) Splice-site utilization. RT-PCR of spliced products transcribed from the different lac repressor constructs produced with the primers indicated in Figure 1. Lanes W_t and S_t contain products amplified from total RNA from testis of animals transgenic for the wtlacI and synlacI transgenes, respectively. All other products are from total RNA of Rat2 cells transfected with the indicated construct. The size of the correctly spliced product (utilizing both the splice donor and splice acceptor sites in the β-actin promoter) is 513 bp for W, 5′C1, and 5′C4; 499 bp for S and 5′C2; and 476 bp in 5′C3 (see Materials and Methods). Note that 5′C1, which contains the wtlacI sequence at the beginning of the coding region, is the only construct other than W to produce predominantly the correctly spliced product. (B) Sequence differences in the region controlling splicing. Bases in the 5′ coding region that differ between wtlacI and synlacI are shown in bold uppercase. The underlined ag indicates the proposed (incorrectly utilized) splice acceptor site in the constructs containing the beginning of the coding region from synlacI. (C,D) Repression of reporter-gene activity by different lac repressor transgenes. Graphs of β-galactosidase reporter-gene activity in Rat2 cells transfected with the regulatable SVOZ construct in combination with the lac repressor construct indicated. Data are expressed as a percentage of the baseline β-galactosidase–expressing cells in the absence of lac repressor. (C) Repressor activity of the parental transgenes (W and S) are shown along with repressor activity of 5′C1 and 5′C2. Note that 5′C1, which is the correctly spliced synlacI construct, does not produce functional repressor activity, indicating that correction of splicing does not restore repressor function. (D) Repressor activity of the 3′ chimeras. Note that those constructs with the wtlacI sequence between the EcoRV and PvuII sites (3′C2 and 3′C4) produce functional repressor, whereas those with the synlacI sequence in this region (3′C1 and 3′C3) do not.

Figure 3

CpG content and gene structure influence transcription of lacI transgenes in the mouse. The lac repressor transgenes that have been used to derive transgenic mice are shown schematically on the right. Each line above the construct map indicates the position of a CpG dinucleotide. The CpG pattern of the endogenous human β-actin promoter and coding region are shown at the top for comparison. Northern blots of 30 μg total RNA from tissues of a mouse transgenic for each of the constructs are shown on the left. The dashed lines highlight two CpG-poor regions in the β-actin locus and their corresponding locations in the lacI transgenes. (B, brain; Th, thymus; H, heart; Lu, lung; Li, liver; Sp, spleen; K, kidney; Te, testis; M, muscle; Sk, skin).

Figure 4

The lac repressor protein is widespread in the LacI^R transgenic mouse. (A) Western blot of total protein from B, brain; H, heart; Lu, lung; Li, liver; S, spleen; K, kidney; T, testis; and M, muscle of a LacI^R transgenic mouse, ntT-testis of a nontransgenic mouse, reacted with a monoclonal antibody raised against the lac repressor. (B–F) Immunohistochemistry with a monoclonal antibody raised against the lac repressor on sections from (B) LacI^R muscle; (C) LacI^R skin; (D) LacI^R hippocampus; (E) LacI^R cerebellum; and (F) nontransgenic cerebellum. Scale bar indicates 500 μm in panels B, E, and F; 200 μm in panels C and D.

Figure 5

Low concentrations of IPTG derepress reporter gene activity in embryonic cells from LacI^R transgenic mice. Embryonic cells were transfected with the SVOZ reporter gene and the number of β-galactosidase–positive cells assayed after growth in varying concentrations of the lactose analog IPTG. (A) Derepression response over the low range of IPTG in primary cells derived from an embryo from the R3 line transgenic for LacI^R. (B) Derepression response on a logarithmic scale of IPTG concentrations for primary cells derived from an embryo from the R1 line transgenic for LacI^R (circles), and a spontaneously immortalized cell line from the primary embryonic R3 cells (squares). Note that all cell lines reach maximal derepression at 0.05 to 0.1 mM IPTG. Both lines tested at higher concentrations (B) showed a decrease in β-galactosidase–positive cells at 20 mM IPTG, which may indicate a toxic level.

Figure 6

IPTG controls LacI^R repression of the Tyr^lacO target gene in the mouse. Mice (A), dissected eyes (B), and cross-sections through eyes (C). Note that the nontransgenic albino and the Tyr^lacO, LacI^R double transgenic lack pigmentation, whereas the Tyrosinase transgenic and the Tyr^lacO, LacI^R double transgenic that has been treated with IPTG are pigmented. (ONL, outer nuclear layer; RPE, retinal pigment epithelium; Scale bar represents 25 μm in C).

Figure 7

Control of the Tyr^lacO target gene is reversible during embryogenesis and after birth. (A) Photomicrographs of embryonic eyes at E12.5. As shown in the far right panel, IPTG can cross the placenta and induce pigmentation in the Tyr^lacO, LacI^R double transgenic RPE, just as in the adult shown in Figure 6. (EC, eye-cup edge; RPE, retinal pigment epithelium; L, lens). (B) Photographs of P3 mice. The corresponding appearance of the eyes at birth is shown for each genotype. The mother of the double-transgenic animal shown in the panel on the far right was started on IPTG at E12.5. The eye pigmentation seen at birth shows that even after the transgene has been silenced by the lac repressor (as demonstrated by the albino phenotype of the Tyr^lacO, LacI^R double-transgenic E12.5 eye in panel A), it can be derepressed by IPTG. (C) Tyrosinase can be silenced after a period of derepression. Left and right panels show the same Tyr^lacO, LacI^R double transgenic animal. On the left as an infant (P8) and on the right as an adult. IPTG was discontinued at P9, causing reversion to an albino phenotype in the adult.