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HT29 cells endogenously express the cystic fibrosis transmembrane conductance regulator (CFTR) and have been used previously as a model to examine cellular regulation of CFTR expression and chloride secretory function. Homologous recombination has been used to specifically disrupt CFTR transcription in the HT29-18-C1 subclone. Experiments demonstrate successful disruption of a CFTR allele by DNA constructs, which target insertion of the neomycin phosphotransferase gene into CFTR exon 1 via homologous recombination. The mutation of one allele is a partial knockout because this cell line has multiple CFTR alleles. The mutation is confirmed by polymerase chain reaction (PCR) and genomic Southern blot analysis. A 52-68% reduction in CFTR mRNA levels is observed in the mutant cell line by both Northern and PCR analysis. However, Western blots show no decrease in total CFTR protein levels. Consistent with the lack of reduction in CFTR protein, the partial knockout mutant does not demonstrate alterations in cyclic AMP or calcium stimulation of chloride efflux or net osmolyte loss. Results suggest that posttranscriptional regulation of CFTR levels may contribute to maintenance of cellular chloride transport function.

Autosomal aneuploidy in mammals adversely affects developmental processes. In human beings, for example, trisomy 21 is the most frequent aneuploidy detected among newborns and the most common known genetic cause of mental retardation. In this review, several hypotheses are discussed that have been proposed to explain the mechanisms by which aneuploidy (especially trisomy) disrupts development. These mechanisms included specific gene dosage effects, generalized disruption of genetic homeostasis, and the influence of the parental origin of the duplicated chromosome. The availability of specific chromosomal rearrangements in mice, coupled with selective breeding schemes, permits generation of aneuploidy of specific chromosomes or chromosomal segments on controlled genetic backgrounds, thus enabling the systematic study of the causes and consequences of defined aneuploidy. Phenotypic characteristics associated with a number of specific aneuploidies in the mouse are discussed. Emphasis is placed on the effects of trisomy 16. Genetic homology between mouse chromosome 16 and human chromosome 21 has led investigators to suggest that analogous mechanisms will be responsible for the developmental abnormalities produced in these respective aneuploidies. Analysis of trisomy 16 mice from the organismal to the subcellular level has revealed a number of phenotypic characteristics (particularly neurobiologic ones) shared with human trisomy 21. The dosage effects of shared genes (or their products) may contribute to the development of these features.

Studies of gene expression during blastocyst formation in mouse preimplantation development have been limited by the amount of RNA available per embryo. Our present approach to this problem has been to construct a large, representative, blastocyst cDNA library in lambda gt11. Random hexadeoxynucleotides were used as primers with total blastocyst RNA serving as template. RNA collected from 4,100 32-64 cell embryos was used to generate a library with an initial size of 30 X 10(6) recombinants. By using clone frequency as a measure of relative mRNA abundance, our data support previous work on the relative and absolute amounts of actin, histone H2a, and intracisternal A particle. Furthermore, we provide estimates for the abundance of cytokeratin endo A, cytokeratin endo B, and beta-tubulin from clone frequency data. Insert sizes for isolated clones range from 200 bp to 3.6 kb with full-length or near-full-length insert sizes for selected clones, indicating that random primer methods generate cDNAs which can represent a significant portion of the mRNA. We have so far characterized products whose abundance is equal to or greater than 0.002% of total RNA. This library offers the potential for the analyses of presumptive regulatory gene products in the mouse preimplantation embryo which are represented as low abundance (less than 1% of mRNA) RNAs.

In this study, we examined the neurochemical profiles of selected brain regions (cerebral hemispheres, diencephalon/brainstem) in fetal (day 14 to 18 gestation) trisomy 19 (Ts19) mice. The neurochemical characteristics we observed in Ts19 mice were quite different from those we observed previously in Ts16 mice. Choline acetyltransferase (ChAT) activity was reduced significantly in the cerebral hemispheres, but not in the brainstem/diencephalon, of the fetal Ts19 mouse brain, suggesting a selective vulnerability of telencephalic cholinergic neurons. Additionally, the activity of glutamic acid decarboxylase (GAD) was reduced significantly in both hemispheres and diencephalon/brainstem of late gestation Ts19 fetuses, suggesting a selective vulnerability of GABAergic neurons as well. While the levels of catecholaminergic and dopaminergic markers were reduced significantly at late gestational ages, the relative rate of turnover of dopamine (DA), measured by the ratio of DOPAC/DA, was elevated significantly in Ts19 mice. Neither reduction in the thickness of various cellular zones of the cerebral cortex nor reduced cell density of the cerebral cortex accounts for the alterations in neurochemical parameters observed in Ts19 mice. These results suggest that the effects of the triplication of specific genes on the respective chromosomes, rather than a generalized disruption of developmental homeostasis resulting from extra chromosomal material, may produce selective alterations in neurochemical and neuroanatomical markers observed in these two mouse trisomies.

We are investigating human insulin gene expression in transgenic mice. An 8.8 kilobase (kb) human genomic DNA fragment, including the insulin gene (1.4 kb) and 2 kb of 5' human flanking sequences, was introduced into mouse embryos by pronuclear microinjection. Two lines of transgenic mice have been established, both of which carry the intact human gene in multiple copies. Animals from both lines have significantly higher insulin levels than control mice, and the degree of hyperinsulinemia shows a positive correlation with human gene copy number in the two lines. Expression of the human gene is confirmed by the detection of human C-peptide in plasma. Tissue specificity of expression is maintained, with human insulin mRNA detectable only in the pancreas. The transgenics maintain normal fasting blood glucose in spite of their high insulin levels, but preliminary studies show them to be glucose intolerant when given a glucose load. These mice provide a model system for further studies on the regulation of insulin gene expression and on the effects of chronic hyperinsulinemia on glucose homeostasis.