Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction

Here we describe a triple transgenic mouse system, which combines the tissue specificity of any Cre-transgenic line with the inducibility of the reverse tetracycline transactivator (rtTA)/tetracycline-responsive element (tet-O)-driven transgenes. To ensure reliable rtTA expression in a broad range of cell types, we have targeted the rtTA transgene into the ROSA26 locus. The rtTA expression, however, is conditional to a Cre recombinase-mediated excision of a STOP region from the ROSA26 locus. We demonstrate the utility of this technology through the inducible expression of the vascular endothelial growth factor (VEGF-A) during embryonic development and postnatally in adult mice. Our results of adult induction recapitulate several different hepatic and immune cell pathological phenotypes associated with increased systemic VEGF-A protein levels. This system will be useful for studying genes in which temporal control of expression is necessary for the discovery of the full spectrum of functions. The presented approach abrogates the need to generate tissue-specific rtTA transgenes for tissues where well-characterized Cre lines already exist.     

Neonatal induction of tolerance to skeletal tissue allografts without immunosuppression

Vascularized allogeneic skeletal tissue transplantation without the need for host immunosuppression would increase reconstructive options for treating congenital and acquired defects. Because the immune system of a fetus or neonate is immature, it may be possible to induce tolerance to allogeneic skeletal tissues by alloantigen injection during this permissive period. Within 12 hours after birth, 17 neonatal Lewis rats were injected through the superficial temporal vein with 3.5 to 5 million Brown Norway bone marrow cells in 0.1 ml normal saline. Ten weeks after the injection, peripheral blood from the Lewis rats was analyzed for the presence of Brown Norway cells to determine hemopoietic chimerism. The Lewis rats then received a heterotopic, vascularized limb tissue transplant (consisting of the knee, the distal femur, the proximal tibia, and the surrounding muscle on a femoral vascular pedicle) from Brown Norway rat donors to determine their tolerance to the allogeneic tissue. A positive control group (n = 6) consisted of syngeneic transplants from Lewis rats into naive Lewis rats to demonstrate survival of transplants. A negative control group (n = 6) consisted of Brown Norway transplants into naive Lewis rats not receiving bone marrow or other immunosuppressive treatment. The animals were assessed for transplant viability 30 days after transplantation using histologic and bone fluorochrome analysis. All the syngeneic controls (Lewis to Lewis) remained viable throughout the experiment, whereas all the Brown Norway to Lewis controls had rejected. Ten of the 17 allografts transplanted into bone marrow recipients were viable at 30 days, with profuse bleeding from the ends of the bone graft and the surrounding graft muscle. The percent of chimerism correlated with survival, with 3.31 percent (SD = 1.9) of peripheral blood, Brown Norway chimerism present in the prolonged survival groups and 0.75 percent (SD = 0.5) of Brown Norway chimerism in the rejected graft group. This study demonstrated prolonged survival of allogeneic skeletal tissue without immunosuppression after early neonatal injection of allogeneic bone marrow in a rat model.     

Autoimmunity and immune complex disease after neonatal induction of transplantation tolerance in mice

Mice made neonatally tolerant to alloantigens were found to develop an immunologic disease resembling systemic lupus erythematosus. In BALB/c mice neonatally injected with C57BL/6 X BALB/c F1 hybrid spleen cells, features of autoimmunity were observed first. After 5-24 wk, antinuclear, anti-SS DNA, thymocytotoxic, and rheumatoid factor-like antibodies were detected in association with hypergammaglobulinemia and with the occurrence of circulating immune complexes and cryoglobulins. Some of the antinuclear antibodies were found to be produced by F1 donor B cells persisting in the host. Second, immunopathologic changes were detected in tolerant mice. In the kidneys, an immune complex glomerulonephritis of the membranous type was observed. Immunoglobulin deposits were also found in the choroid plexus and at the dermoepidermal junction. In addition, thrombocytopenia was a common finding, and a positive direct Coomb's test occasionally was detected. Features of autoimmune disease were closely associated with the effective induction of transplantation tolerance, as revealed by the inability of spleen cells to generate in vitro cytolytic responses against C57BL/6 alloantigens. It is suggested that, although transplantation tolerance is associated with a lack of cytolytic reaction of the host against F1 hybrid donor alloantigens, other types of allogeneic interactions could lead in this model to the development of autoimmunity and immunopathology.     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. IV. Significance of intrathymic chimerism of blood-born Ia+ cells in Mls tolerance.

The significance of thymus cell chimerism in the induction and maintenance of tolerance was investigated. Mls-1b BALB/c mice were neonatally tolerized by the intravenous administration of either bone marrow (BM) cells or peritoneal cavity (PerC) cells from Mls-1b/a (BALB/c x AKR) F1 mice. Tolerance was long-lasting in the BM cell group, but transient in the PerC cell group, probably because PerC cells lack hemopoietic stem cells required for a continuous supply of tolerance-inducing cells. The degree of anti-Mls-1a responsiveness of these BALB/c thymus cells was correlated with the degree of intrathymic distribution of the inoculated F1 cells. The effect of BM cell inoculation, resulting in a year-long deletion of Mls-1a-reactive V beta 6-bearing T cells is in marked contrast to that of PerC cell inoculation which causes only a transient loss of V beta 6+ mature thymocytes (for about 1 week after birth). This functional profile of the tolerant state correlates well with the degree and persistence of the intrathymic presence of F1 type Ia+ cells. The long-lasting presence of donor-derived cells throughout the thymus tissue in the BM cell group is also in marked contrast to the early disappearance of Ia+ cells (within 2-3 weeks) from the cortex and then from the medulla in the PerC cell group, although these Ia+ cells were once spread throughout the thymus tissue 4 days after the tolerance-inducing cell inoculation. Taken together with a failure to induce consistent unresponsiveness to Mls-1a determinants in Mls-1b thymocytes regenerating in Mls-1a-thymic epithelial environments, all the above data indicate that intrathymic chimerism caused by hemopoietic stem cell-derived MHC-class II-bearing cells is a requisite for the induction and maintenance of unresponsiveness by means of clonal deletion in experimentally as well as naturally induced tolerance to Mls determinants.     

Pharmacological-based translational induction of transgene expression in mammalian cells

In the quest for the development of pharmacological switches that control gene expression, no system has been reported that regulates at the translational level. To permit small-molecule control of transgene translation, we have constructed a farnesyl transferase inhibitor-responsive translation initiation factor. This artificial protein is a three-component chimaera consisting of the ribosome recruitment core of the eIF4G1 eukaryotic translation initiation factor, the RNA-binding domain of the R17 bacteriophage coat protein and the plasma membrane localization CAAX motif of farnesylated H-Ras. This membrane-delocalized translation factor is inactive unless liberated in the cytosol. Farnesyl transferase inhibitor FTI-277 prevents the membrane association of the CAAX motif and thus increases the cytoplasmic levels of the eIF4G fusion protein, which is then capable of inducing translation of the second cistron of a bicistronic messenger RNA containing an R17-binding site in its intercistronic space. Such direct translational control by farnesyl transferase inhibitors provides a system for fast, graded and reversible regulation of transgene expression.     

Effects of in vivo monoclonal anti-I-A antibody treatment in neonatal mice on intrathymic and peripheral class II antigen expression

Intrathymic, Ia-bearing antigen-presenting cells (APC) are believed to play an important role in the development of a mature, functional T-cell repertoire. We used chronic in vivo treatment of neonatal mice with anti-I-A monoclonal Ab (MAb) to examine the expression of I-A and I-E antigens on intrathymic and peripheral APC. Three weeks after continuous treatment with anti-I-A MAb, FACS analysis of unfractionated spleen cells revealed a 75-90% reduction in the number of I-A bearing cells. Splenic antigen-presenting capacity measured by the ability of unseparated or density gradient-enriched APC to stimulate I-A- or I-E-reactive T-cell hybridomas was also greatly reduced. In contrast to the expression of I-A and I-E molecules in the splenic APC, anti-I-A MAb treatment resulted in decreased thymic APC I-A expression without significant changes in I-E as measured by FACS analysis. This was confirmed in functional studies in which allo-I-A- or auto-I-A-reactive T-cell hybridomas could not be stimulated by treated thymic APC. Unlike splenic APC, anti-I-A-treated thymic APC did not differ significantly from normals in their ability to stimulate allo-I-E-reactive T hybridomas. This lack of linkage or comodulation of I-A and I-E expression on thymic but not splenic APC may allow us to study the role of I-A molecules and I-E molecules on the development and expansion of functional, mature T-cell repertoires.     

Analysis of early events occurring during neonatal induction of tolerance to histoincompatible cells in mice

Mice have been analyzed at different times post neonatal inoculation of F₁ hybrid lymphoid cells for their ability to respond to foreign MHC antigens expressed on the tolerizing cell population, and to impart that response phenotype to normal adult spleen cells. At early times following neonatal challenge with F₁ spleen cells all mice produced serum immunoglobulins of F₁ donor origin which inhibited the response of normal adult cells. The antigenic specificity of the inhibitory factors suggests a role for an antireceptor antibody in the inhibition seen, and the presence of such serum-mediated inhibition is indicative of (and may be causally related to) the late appearance of a cell-mediated suppressor mechanism in these mice.     

Recombinant human granulocyte colony-stimulating factor improves the compromised state of recipient mice without affecting the induction of specific tolerance in the cyclophosphamide-induced tolerance system.

The effects of recombinant human granulocyte CSF (rhG-CSF) on cyclophosphamide (CP)-induced tolerance was studied. In the recipient C57BL/10 Sn Slc (B10) mice given 1 x 10(8) B10.BR Sg Sn Slc (B10.BR) spleen cells (SC) on day -2 followed by 200 mg/kg CP on day 0, the number of leukocytes and neutrophils in the periphery declined to their minimum levels on day 4. When rhG-CSF in a dose of 200 micrograms/kg was given daily for 5 days to the B10 mice, which had been treated with B10.BR SC and CP, starting one day after the administration of CP, the leukocyte and neutrophil counts declined to the same levels as those in the B10 mice treated with B10.BR SC and CP alone on day 2. On day 4, however, the counts recovered to their normal levels. The nucleated cell count of the spleen in the B10 mice given B10.BR SC and CP followed by rhG-CSF decreased less and recovered faster than that in the B10 mice given B10.BR SC and CP. The case was found to be the same in bone marrow, and the difference did not reach statistical significance. When the recipient mice were inoculated i.p. with 4 x 10(4) Pseudomonas aeruginosa (GNB-139) on day 4, the survival of the B10 mice treated with B10.BR SC and CP was markedly improved by rhG-CSF administration. The administration of rhG-CSF did not affect either the prolongation or the specificity of skin allograft survival, as shown in an H-2 mis-matched combination of B10.BR----B10 and in an H-2 identical combination of AKR/J Sea(AKR)----C3H/HeN Crj (C3H). The tolerant state, which was demonstrated by various immune responses, such as CTL, delayed footpad reaction, and antibody, was also not affected by rhG-CSF. Furthermore, the basic mechanisms for inducing a long-lasting skin allograft tolerance in this system--namely, the specific destruction of Ag-stimulated and then proliferating mature T cells in the periphery, the establishment of mixed chimerism, and the intrathymic clonal deletion of immature T cells--were preserved even when rhG-CSF was given to C3H mice previously made tolerant of AKR.     

Effect of the E4 region on the persistence of transgene expression from adenovirus vectors.

The utility of adenovirus vectors for gene therapy is limited by the transience of expression that has been observed in various in vivo models. Immunological responses to viral targets can eliminate transduced cells and cause the loss of transgene expression. We previously described the characterization of an E4 modified adenovirus, Ad2E4ORF6, which is replication defective in cotton rats. We reasoned that gene transfer vectors based on Ad2E4ORF6 would have a reduced potential for viral gene expression in vivo which might be beneficial for achieving persistence of transgene expression. E1 replacement vectors expressing the cystic fibrosis transmembrane regulator or beta-galactosidase were constructed as series of vectors that differed with respect to the E4 region. Vectors containing a wild-type E4 region, E4 open reading frame 6, or a complete E4 deletion were compared in the lungs of BALB/c mice for persistence of expression. Results obtained with nude mice indicate that nonimmunological factors have a major influence on the longevity of transgene expression. Expression was transient from the E1a promoter with all vectors but persisted from the cytomegalovirus promoter only with a vector containing a wild-type E4 region. Transience of expression did not correlate with the disappearance of vector DNA, suggesting that promoter down-regulation may be involved. Coinfection studies indicate an E4 product(s) could be supplied in trans to allow persistent expression from the cytomegalovirus promoter. In summary, the choice of promoter is important for achieving persistence of expression; in addition, some promoters are highly influenced by the context of the vector backbone.     

Activation of transgene expression by early region 4 is responsible for a high level of persistent transgene expression from adenovirus vectors in vivo.

The persistence of transgene expression has become a hallmark for adenovirus vector evaluation in vivo. Although not all therapeutic benefit in gene therapy is reliant on long-term transgene expression, it is assumed that the treatment of chronic diseases will require significant persistence of expression. To understand the mechanisms involved in transgene persistence, a number of adenovirus vectors were evaluated in vivo in different strains of mice. Interestingly, the rate of vector genome clearance was not altered by the complete deletion of early region 4 (E4) in our vectors. The GV11 (E1- E4-) vector genome cleared with a similar kinetic profile as the GV10 (E1-) vector genome in immunocompetent and immunocompromised mice. These results suggest that the majority of adenovirus vector genomes are eliminated from transduced tissue via a mechanism(s) independent of T-cell, B-cell, and NK cell immune mechanisms. While the levels of persistence of transgene expression in liver or lung transduced with GV10 and GV11 vectors expressing beta-galactosidase, cystic fibrosis transmembrane conductance regulator, or secretory alkaline phosphatase were similar in immunocompetent mice, a marked difference was observed in immunocompromised animals. Levels of transgene expression initially from both GV10 and GV11 vectors were the same. However, GV11 transgene expression correlated with loss of vector genome, while GV10 transgene expression persisted at a high level. Coadministration and readministration of GV10 vectors showed that E4 provided in trans could activate transgene expression from the GV11 vector genome. While transgene expression activity per genome from the GV10 vector is clearly activated, expression from a cytomegalovirus promoter expression cassette in a GV11 vector appeared to be further inactivated as a function of time. Understanding the molecular mechanisms underlying these expression effects will be important for developing persistent adenovirus vectors for chronic applications.     

The use of haptenated-immunoglobulin molecules to induce tolerance in B cells from neonatal mice

The role of the Fc region of trinitrophenylated (TNP)-immunoglobulins (Ig) in their ability to induce tolerance in immature B cells was examined. With the use of B cells from neonatal mice, tolerogens that could or could not bind to Fc receptors were assessed for their ability to induce tolerance. This was accomplished by tolerizing spleen cells in bulk culture and assessing the degree of tolerance by challenging the cells with the thymus-independent antigen TNP-Brucella abortus (TNP-BA) in limiting dilution cultures. It was found that by using tolerogens containing 10 to 11 haptens per Ig molecule, immature B cells were very susceptible to tolerance induction. Mature B cells were not as susceptible. This increased susceptibility was independent of the Fc portion of the tolerogen, because TNP11-HGG and a TNP10-F(ab')2 induced equivalent degrees of unresponsiveness. When the TNP density was lowered to approximately five haptens per Ig molecule, those Ig molecules that contained Fc portions were superior tolerogens with the use of B cells from 6-day-old mice. Thus, a TNP4-HGG, TNP7-mouse IgG1, and TNP6-mouse IgG2a were more effective tolerogens than either TNP5-F(ab')2 or TNP6-mouse IgG3. These results confirm previous findings that immature B cells are inherently more susceptible to tolerance induction than mature B cells. They also suggest that very lightly haptenated Ig molecules may depend on Fc receptor-binding for effective tolerance induction. Finally, by means of a cytofluorograph, the surface IgD (sIgD) and sIgM phenotypes of splenic B cells from neonates of increasing age were determined. When comparing the phenotype of maturing cells with their tolerance susceptibilities, a correlation between the appearance of sIgD and the acquisition of resistance to tolerance was observed.     

Epidermal expression of an Elovl4 transgene rescues neonatal lethality of homozygous Stargardt disease-3 mice

Elongase of very long chain fatty acids-4 (ELOVL4) is the only mammalian enzyme known to synthesize C28-C36 fatty acids. In humans, ELOVL4 mutations cause Stargardt disease-3 (STGD3), a juvenile dominant macular degeneration. Heterozygous Stgd3 mice that carry a pathogenic mutation in the mouse Elovl4 gene demonstrate reduced levels of retinal C28-C36 acyl phosphatidylcholines (PC) and epidermal C28-C36 acylceramides. Homozygous Stgd3 mice die shortly after birth with signs of disrupted skin barrier function. In this study, we report generation of transgenic (Tg) mice with targeted Elovl4 expression driven by an epidermal-specific involucrin promoter. In homozygous Stgd3 mice, this transgene reinstates both epidermal Elovl4 expression and synthesis of two missing epidermal lipid groups: C28-C36 acylceramides and (O-linoleoyl)-omega-hydroxy C28-C36 fatty acids. Transgene expression also restores skin barrier function and rescues the neonatal lethality of homozygous Stgd3 mice. These studies establish the critical requirement for epidermal C28-C36 fatty acid synthesis for animal viability. In addition to the skin, Elovl4 is also expressed in other tissues, including the retina, brain, and testes. Thus, these mice will facilitate future studies to define the roles of C28-C36 fatty acids in the Elovl4-expressing tissues.     

Genetic control of tolerance induction to human gamma-globulin in mice.

Susceptibility to tolerance induction with monomeric human gamma-globulin (HGG) was tested in different inbred strains of mice. The results indicated a differential tolerance susceptibility among the strains and that the basis for the variation is genetic in nature. By using a protocol that permits genetic analysis, F1, F2, and backcross generations of the parental strains SJL/J and C3H/Bi were examined. A multigenic control model by H-2-linked and non-H-2-linked genes showing Mendelian autosomal inheritance is proposed.     

Long-lasting consequences of neonatal maternal separation on social behaviors in ovariectomized female mice

Maternal separation (MS) stress is known to induce long-lasting alterations in emotional and anxiety-related behaviors, but effects on social behaviors are not well defined. The present study examined MS effects on female social behaviors in the social investigation (SIT) and social preference (SPT) tests, in addition to non-social behaviors in the open-field (OFT) and light-dark transition (LDT) tests in C57BL/6J mice. All females were tested as ovariectomized to eliminate confounding effects of endogenous estrogen during behavioral testing. Daily MS (3 hr) from postnatal day 1 to 14 did not affect anxiety levels in LDT, but were elevated in OFT with modified behavioral responses to the novel environment. Furthermore, MS altered social investigative behaviors and preference patterns toward unfamiliar stimulus mice in SIT and short- and long-term SPT paradigms. In SIT, MS reduced social investigation duration and increased number of stretched approaches towards both female and male unfamiliar stimulus mice, suggesting increased social anxiety levels in MS females. Similarly, MS heightened levels of social anxiety during short-term SPT but no MS effect on social preference was found. On the other hand, MS females displayed a distinctive preference for female stimuli, unlike control females, when tested for long-term SPT over a prolonged period of 5 days. Evaluation of FosB expression in the paraventricular nucleus, medial and central amygdala following stimulus exposure demonstrated greater number of FosB immunopositive cells in all three brain regions in MS females compared to control females. These results suggest that MS females might differ in neuroendocrine responses toward unfamiliar female and male opponents, which may be associated with modifications in social behaviors found in the present study. Taken together, this study provides new evidence that early life stress modifies female social behaviors by highlighting alterations in behavioral responses to situations involving social as well as non-social novelty.     

B- and T-cell tolerance induction in young-adult and old mice.

Young-adult and old (CS7BL × C3H)F₁ mice were injected with either of two tolerogens followed by challenge with dinitrophenylated human γ globulin (DNP-HGG). The primary IgM and IgG responses of spleen cells to the DNP determinant were evaluated by a modified hemolytic plaque assay. Carrier-specific thymus-derived cell (T-cell) tolerance was induced in mice with deaggregated HGG. Hapten-specific bursal-derived cell (B-cell) tolerance was induced with DNP coupled to isogeneic mouse IgG. The dose of these two tolerogens was successively decreased by 10-fold decrements until a response similar to that of control mice was achieved. The minimum tolerizing dose (MTD) was then determined for young-adult and aged B-and T-cells. We found that the MTD for old B-cells was 10 times greater than that obtained with young B-cells for both the direct and indirect PFC responses. No difference in MTD was observed between young and old T-cells when assessed by the indirect response; the MTD for old T-cells was 10-fold greater than that obtained for young-adult T-cells when the direct response was evaluated. A double threshold of tolerance was found for T-cells of young-adult mice.