Multiple regulatory domains in the mouse mammary tumor virus long terminal repeat revealed by analysis of fusion genes in transgenic mice.

Transcription initiated within the mouse mammary tumor virus (MTV) long terminal repeat (LTR) is regulated by glucocorticoids, androgens, and estrogen. However, expression of the virus in vivo and transcription of MTV LTR fusion genes in transgenic mice are not readily interpretable solely in terms of the influence of these hormones. To investigate whether there is a regulatory role for sequences within the LTR but outside the region known to be responsible for glucocorticoid induction, we have produced transgenic mice carrying genes in which various regions of the LTR have been linked to the human growth hormone gene. Analysis of expression of the fusion genes in these transgenic mice has demonstrated that the 5' end of the LTR can profoundly influence transcription initiated within the MTV LTR.

Osteoblast-specific expression of growth hormone stimulates bone growth in transgenic mice.

Growth hormone (GH) is an important regulator of postnatal growth, acting on a wide variety of target tissues. Here, we show that local production of GH in osteoblasts is able to stimulate bone growth directly without significant systemic effects. Mice were made transgenic by microinjection of an osteocalcin-human GH (osteocalcin-hGH) gene construct in which approximately 1,800 bp of the rat osteocalcin promoter was fused to the hGH gene. Five lines of transgenic mice, each with measurable amounts of serum hGH (ranging from 1 to 1,000 ng/ml), were analyzed. Northern (RNA) blot hybridization showed that the hGH transcript was detectable only in the bone. Further characterization of hGH mRNA distribution by in situ hybridization revealed that in neonates the most intense signal was found in periosteal osteoblasts, while in adults, trabecular and endosteal osteoblasts were favored. In one transgenic line (992-1), hGH was expressed at a much lower level and had minimal systemic effects; however, the local concentrations of hGH in bone were sufficient to stimulate bone growth in these animals.

Haploid-specific transcription of protamine-myc and protamine-T-antigen fusion genes in transgenic mice.

The protamines are small, basic, arginine-rich proteins synthesized postmeiotically in the testes. Analysis of the regulation of synthesis of the protamine mRNA and protein is restricted by the difficulty in culturing and manipulating the cells in which transcription and translation occur. To avoid these problems, we have produced transgenic mice carrying fusion genes in which sequences 5' to the mouse protamine-2 gene have been linked to exons 2 and 3 of the mouse c-myc gene and, separately, to the simian virus 40 (SV40) early region. We show here that the prot.myc gene is correctly regulated; transcription is detected only in the round spermatids. In one family of transgenic mice carrying the 5' protamine-SV40 T-antigen fusion gene, SV40 early-region mRNA accumulated to the highest level in the testes but was also detected in the thymuses, brains, hearts, and preputial glands of the animals. Although we have demonstrated specific transcription of these fusion genes in the round spermatids, we were not able to detect the SV40 T-antigen protein.

Overexpression of the retinoic acid-responsive gene Stra6 in human cancers and its synergistic induction by Wnt-1 and retinoic acid.

Genetic defects in the Wnt-1 signaling pathway contribute to human tumor progression and are especially prevalent in colorectal cancer. We screened mouse C57MG cells to isolate mRNAs induced by Wnt-1 and identified Stra6, an mRNA known to be up-regulated by retinoic acid. Up-regulation of Stra6 mRNA was also observed in hyperplastic mammary tissue and mammary gland tumors from transgenic mice expressing Wnt-1 and in human tumors that frequently harbor defects in Wnt-1 signaling. Stimulation of C57MG cells with retinoic acid plus Wnt-1 resulted in expression of Stra6 transcript to levels greatly exceeding that observed with either stimulus alone. This synergy could be explained in part by the up-regulation of retinoic acid receptor-gamma that was observed in response to Wnt-1 signaling. Accordingly, treatment of human colorectal cancer cell lines with retinoic acid resulted in the up-regulation of Stra6 mRNA and accumulation of Stra6 protein at the cell membrane. The data support a model in which Wnt-1 signaling synergizes with retinoids to activate retinoic acid receptor-gamma-responsive genes in human cancers.

A dominant phenocopy of hypopituitarism in transgenic mice resulting from central nervous system synthesis of human growth hormone.

We have produced a line of transgenic mice in which expression of human GH has been detected only in the cerebral cortex. Both male and female transgenic mice are growth inhibited with respect to their nontransgenic littermates. Mouse GH mRNA and insulin-like growth factor-I mRNA levels in the pituitary and liver, respectively, are reduced, and circulating insulin-like growth factor-I levels are lower in these mice. Within the hypothalamus somatostatin mRNA levels are increased and GH-releasing factor mRNA levels are reduced compared to those in nontransgenic littermates. We suggest that the growth retardation in these mice is a consequence of the ectopic human GH disturbing the normal controls that regulate mouse GH synthesis and release from the pituitary. These mice provide a resource for analysis of the regulation of GH production and demonstrate that a dominant phenocopy can be made by producing transgenic mice that have local production of an extra-cellular hormone.

The aza-arenes as mutagens for Salmonella typhimurium.

Several unsubstituted aza-arenes have been found to be more mutagenic to Salmonella typhimurium than their corresponding parent hydrocarbons. In most cases, the activity of these compounds depended on the presence of a post-mitochondrial supernatant for metabolic activation, although acridine was mutagenic only in the absence of such an activating system. An examination of the effect of the metabolizing system's concentration on mutagenicity showed that quinoline, benzo[f]quinoline, and phenanthridine have different optima. In an attempt to uncover active intermediates in aza-arene metabolism, N-oxides of quinoline and phenanthridine were synthesized and found to be non-mutagenic, and coincubation with the epoxide hydrase inhibitor trichloropropylene oxide did not affect the mutagenic activity of quinoline or phenanthridine.

St. Louis encephalitis virus temperature-sensitive mutants. I. Induction, isolation and preliminary characterization.

Nine temperature-sensitive (ts) mutants of St. Louis encephalitis virus were isolated after "forced mutagenesis" with 5-fluorouracil or 5-azacytidine. The ts mutants could be grouped on the basis of RNA synthesis at 40 degrees C, the nonpermissive temperature and complementation analysis. Four complementation groups were identified. Members of two of the groups were negative for RNA synthesis at 40 degrees C while the remainder were positive.

Transgenic mice as a model to test the immunogenicity of proteins altered by site-specific mutagenesis.

Second generation therapeutic proteins are now being produced by in vitro mutagenesis of the relevant genes. Of some concern, however, is the possibility that these altered proteins will be immunogenic and the antibodies raised will also recognize the endogenous protein with undesirable consequences. We have designed a biological system to test these possibilities. In this model, transgenic mice produce and secrete human tissue plasminogen activator (h.tPA) to which these mice are immunologically tolerant. However, when challenged with a form in which a single amino acid has been substituted these mice will produce antibodies capable of recognizing h.tPA. These results indicate that there are immunological consequences connected with the design and administration of second generation therapeutic proteins.