Ubiquitous expression of marker transgenes in mice and rats.

The ability to unambiguously mark a cell's genotype is essential for studies in which genetically distinct cell populations must be distinguished from one another in vivo. One approach to this challenge has been the creation of transgenic mice expressing a transgene marker that is easily detectable, with no background staining. Multiple transgenic mouse strains bearing constructs with different combinations of promoter elements and coding sequences have been described, each with its own advantages and limitations. In this report we describe the use of an 800-bp promoter fragment isolated from the beta(geo) integration site in ROSA26 mice to target expression of two marker genes. We demonstrate that the ROSA26 promoter directs ubiquitous expression of human placental alkaline phosphatase and enhanced green fluorescent protein during embryonic and postnatal development in mouse and rat. We further demonstrate the general utility of these transgenes for marking donor cells in transplantation studies.

Transgenic hepatocarcinogenesis in the rat.

Although transgenic hepatocarcinogenesis has been accomplished in the mouse with a number of genetic constructs targeting the oncogene to expression primarily in the liver, no example of this process has yet been developed in the rat. Because our understanding of the multistage nature of hepatocarcinogenesis is most advanced in the rat, we have developed a strain of transgenic rats carrying the promoter-enhancer sequences of the mouse albumin gene linked 5' to the simian virus-40 T antigen gene. A line of transgenic rats bearing this transgene has been developed from a single founder female. Five to six copies of the transgene, possibly in tandem, occur within the genome of the transgenic animals, which are maintained by heterozygous matings. Livers of transgenic animals are histologically normal after weaning; at 2 months of age, small foci of vacuolated cells appear in this organ. By 4 months of age, all animals exhibit focal lesions and nodules consisting primarily of small basophilic cells, many of which exhibit considerable cytoplasmic vacuolization. Mating of animals each bearing the transgene results in rats with a demyelinating condition that develops acutely in pregnant females and more chronically in males. Ultrastructural studies of these cells indicate that the vacuoles contain substantial amounts of glycogen, with the cells resembling hepatoblasts. Malignant neoplasms with both a glandular and a hepatoblastoma/hepatocellular carcinoma pattern arise from the nodules. Enzyme and immunohistochemical studies of all lesions reveal many similarities in gene expression to comparable lesions in rats subjected to chemically induced hepatocarcinogenesis, with certain exceptions. The placental form of glutathione-S-transferase is absent from all lesions in the transgenic animal, as is the expression of connexin 32. A significant number of lesions express serum albumin, and many, but not all, exhibit the T antigen. Lesions expressing the T antigen also contain stainable amounts of the p53 gene product; by contrast, normal hepatocytes express only very low levels of the T antigen within their nuclei and no demonstrable p53. All of the animals develop hepatic lesions, and approximately one-third also develop adenomas and carcinomas derived from the islet cells of the pancreas. Although there are differences in the morphology, biology, and genetic expression in early and late hepatic lesions in this strain of transgenic rat, many similarities also occur, making this a potential model system with which to study the interactions of environmental factors with a genetic program for hepatocarcinogenesis.

Tumorigenicity by human papillomavirus type 16 E6 and E7 in transgenic mice correlates with alterations in epithelial cell growth and differentiation.

The human papillomavirus type 16 (HPV-16) E6 and E7 oncogenes are thought to play a role in the development of most human cervical cancers. These E6 and E7 oncoproteins affect cell growth control at least in part through their association with and inactivation of the cellular tumor suppressor gene products, p53 and Rb. To study the biological activities of the HPV-16 E6 and E7 genes in epithelial cells in vivo, transgenic mice were generated in which expression of E6 and E7 was targeted to the ocular lens. Expression of the transgenes correlated with bilateral microphthalmia and cataracts (100% penetrance) resulting from an efficient impairment of lens fiber cell differentiation and coincident induction of cell proliferation. Lens tumors formed in 40% of adult mice from the mouse lineage with the highest level of E6 and E7 expression. Additionally, when lens cells from neonatal transgenic animals were placed in tissue culture, immortalized cell populations grew out and acquired a tumorigenic phenotype with continuous passage. These observations indicate that genetic changes in addition to the transgenes are likely necessary for tumor formation. These transgenic mice and cell lines provide the basis for further studies into the mechanism of action of E6 and E7 in eliciting the observed pathology and into the genetic alterations required for HPV-16-associated tumor progression.

Chondroitin sulfate facilitates an acrosome reaction in bovine spermatozoa as evidenced by light microscopy, electron microscopy and in vitro fertilization.

Bovine epididymal spermatozoa were incubated for 22 h in a modified Tyrode's medium. The percentages of sperm exhibiting an acrosome reaction were determined morphologically after fixing and staining specimens. The addition of chondroitin sulfate A (CS-A) significantly increased the incidence of acrosome reaction. When observed by electron microscopy, acrosome-reacted sperm had undergone vesiculation of the plasma and outer acrosomal membranes. Sperm incubated in the presence of CS-A demonstrated a significantly higher incidence of vesiculation when compared to the controls. Additionally, rates of in vitro fertilization of bovine oocytes were significantly elevated when sperm and ova were exposed to CS-A. These results suggest that glycosaminoglycans in the female reproductive tract may be responsible for some of the biochemical changes associated with fertilization, and a light microscope procedure can be used to assess occurrence of the acrosome reaction.