Effects In Transgenic Mice Research Articles

Modification of Bacterial Artificial Chromosomes (BACs) and Preparation of Intact BACDNA for Generation of Transgenic Mice

BAC transgenesis is a powerful tool for the study of gene expression and gene function in the mouse in vivo. In this unit, detailed protocols are provided for modification (i.e., marker gene insertion, deletion, or point mutation) of BACs by homologous recombination in E. coli. This method utilizes a shuttle vector that allows transient expression of the E. coli RecA gene to support homologous recombination in the BAC host bacteria. In addition, two protocols are provided for purification of BAC DNA for microinjection to generate transgenic mice. Since BAC DNA is prone to degradation, which may introduce positional effects in transgenic mice, two methods are given for purification of intact BAC DNA for subsequent microinjection.

Augmented systolic response to the calcium sensitizer EMD-57033 in a transgenic model with troponin I truncation

Myocardial stunning is a form of acute reversible cardiac dysfunction that occurs after brief periods of ischemia and reperfusion. In several animal models, stunning is associated with proteolytic truncation of troponin I (TnI). Mice expressing the same proteolytic TnI fragment [TnI-(1-193)] demonstrate cardiac depression with a decreased maximal calcium-activated tension. We therefore hypothesized preferential improvement in mice expressing TnI-(1-193) treated with the calcium-sensitizing drug EMD-57033. TnI-(1-193) and nontransgenic myofibrils exhibited significant sensitization to calcium in Mg-ATPase assays after EMD-57033 exposure. However, only transgenic myofibrils exhibited an increase in maximal activity (P = 0.023). EMD-57033 also increased maximal calcium-activated force in TnI-(1-193) muscle, such that it was comparable to nontransgenic cardiac muscle. EMD-57033 enhanced in vivo systolic function modestly in controls but had a marked effect in transgenic mice, with an almost threefold greater leftward shift of the end-systolic pressure-volume relation (P = 0.0005). These data indicate a targeted efficacy of EMD-57033 in offsetting the contractile defect in TnI-(1-193) mice, and this may have therapeutic implications in models displaying this myofilament defect.

Evidence that DNase I hypersensitive site 5 of the human beta-globin locus control region functions as a chromosomal insulator in transgenic mice.

We have previously reported that DNase I hypersensitive site 5 (5'HS5) of the human beta-globin locus control region functions as a chromatin insulator in stable transfection assays. In this report we show that a 3.2 kb DNA fragment containing the entire 5'HS5 region can protect a position-sensitive (A)gamma-globin gene against position effects in transgenic mice. Bracketing is required for function of 5'HS5 as an insulator. The 5'HS5 insulator operates in adult as well as in embryonic murine erythroid cells. The insulator has no significant stimulatory effects of its own. These results indicate that 5'HS5 can function as a chromatin insulator in vivo.

Cooperation and competition between the binding of COUP-TFII and NF-Y on human epsilon- and gamma-globin gene promoters.

The nuclear receptor COUP-TFII was recently shown to bind to the promoter of the epsilon- and gamma-globin genes and was identified as the nuclear factor NF-E3. Transgenic experiments and genetic evidence from humans affected with hereditary persistence of fetal hemoglobin suggest that NF-E3 may be a repressor of adult epsilon and gamma expression. We show that, on the epsilon-promoter, recombinant COUP-TFII binds to two sites, the more downstream of which overlaps with an NF-Y binding CCAAT box. Binding occurs efficiently to either the 5' or the 3' COUP-TFII site but not to both sites simultaneously. However, adding recombinant NF-Y induces the formation of a stable COUP-TFII.NF-Y-promoter complex at concentrations of COUP-TFII that would not give significant binding in the absence of NF-Y. Mutations of the promoter indicate that COUP-TFII cooperates with NF-Y when bound to the 5' site, whereas binding at the 3' site is mutually exclusive. Likewise, in the gamma-promoter, COUP-TFII binds to a site overlapping the distal member of a duplicated CCAAT box, competing with NF-Y binding. Transfections in K562 cells show that both the mutation of the 5' COUP-TFII or of the NF-Y site on the epsilon-promoter decrease the activity of a luciferase reporter; the mutation of the 3' COUP-TFII site has little effect. These results, together with transgenic experiments suggesting a repressive activity of COUP-TFII on the epsilon-promoter and the observation that, on the 3' site, COUP-TFII and NF-Y binding is mutually exclusive, suggest that COUP-TFII may exert different effects on epsilon transcription depending on whether it binds to the 5' or to the 3' site. At the 5' site, COUP-TFII might cooperate with NF-Y, forming a stable complex, and stimulate transcription; at the 3' site, COUP-TFII might compete for binding with NF-Y and, directly or indirectly, decrease gene activity.

Adipocyte Insensitivity to Insulin in Growth Hormone-Transgenic Mice

Growth hormone (GH) has an inhibitory effect on adipogenesis, and its effect is associated with insulin action in obesity. In this study, the relationship between GH effect on insulin sensitivity and adipocyte differentiation in vivo was investigated. Transgenic (TG) female mice expressing porcine GH had reduced body weights and weights of retroperitoneal and parametrial fat depots. Insulin treatment increased PPARγ and GLUT4 expression in adipose tissue of WT mice but had no effect in TG mice. Content of transcription factors, PPARγ and C/EBPα and β, was higher in adipose tissue of WT mice, and for C/EBPα and PPARγ, the difference occurred primarily in 24-, compared to 12-week-old, mice. Expression of preadipocyte factor-1 was higher in adipose tissue of TG mice, and expression of TNF-α and leptin was reduced in adipose tissue of 24-week-old TG mice. Our results suggest that increased expression of GH reduces adipogenesis by inducing adipocyte resistance to the adipogenic effect of insulin.

A globin enhancer acts by increasing the proportion of erythrocytes expressing a linked transgene.

Enhancer elements have been shown to affect the probability of a gene establishing an active transcriptional state and suppress the silencing of reporter genes in cell lines, but their effect in transgenic mice has been obscured by the use of assays that do not assess expression on a cell-by-cell basis. We have examined the effect of a globin enhancer on the variegation of lacZ expression in erythrocytes of transgenic mice. Mice carrying lacZ driven by the alpha-globin promoter exhibit beta-galactosidase (beta-Gal) expression in only a very small proportion of embryonic erythrocytes. When the transgenic construct also contains the (alphaHS-40 enhancer, which controls expression of the alpha-globin gene, expression is seen in a high proportion of embryonic erythrocytes, although there are variations between transgenic lines which can be attributed to different sites of integration. Analysis of beta-Gal expression levels suggests that expressing cells in lines carrying only the alpha-globin promoter express as much beta-Gal as those in which the transgene also contains alphaHS-40. A marked decline in transgene expression occurs as mice age, which is mainly due to a decrease in the proportion of cells expressing the transgene. Thus, a globin enhancer can act to suppress variegation of a linked transgene; this result is consistent with a model in which enhancers act to establish and maintain an active domain without directly affecting the transcriptional rate.

High level overexpression of glucose transporter-4 driven by an adipose-specific promoter is maintained in transgenic mice on a high fat diet, but does not prevent impaired glucose tolerance.

High fat feeding is associated with impaired insulin action, an obese body composition, and down-regulation of glucose transporter-4 (GLUT4) expression in adipocytes. We recently showed that overexpression of GLUT4 selectively in adipocytes of transgenic mice using the aP2 (fatty acid-binding protein) promoter/enhancer results in enhanced glucose tolerance and adipocyte hyperplasia. Here, we fed these GLUT4-overexpressing transgenic mice a high fat (55%) or a low fat (10%) diet for 13-15 weeks to determine the role of alterations in GLUT4 expression in adipocytes in the development of insulin resistance and obesity, which are characteristic of high fat consumption. In nontransgenic mice, high fat feeding results in 45-50% reduction of GLUT4 levels in white and brown adipose tissue, with a parallel decrease in insulin-stimulated glucose transport. In transgenic mice receiving the low fat diet, GLUT4 is overexpressed 20-fold in white and 4-fold in brown adipose tissue. Glucose transport in epididymal adipocytes is increased 20-fold in the basal state and 6-fold in the insulin-stimulated state. Even after transgenic mice are fed a high fat diet, GLUT4 expression and glucose transport in their adipocytes remains 14- to 30-fold greater than that in nontransgenic mice receiving the same diet. Despite these marked effects at the adipose cell level, glucose tolerance is not improved, probably due to insulin resistance in skeletal muscle and liver, where the transgene is not expressed. During the low fat diet, transgenic mice have 80% more body lipid than nontransgenics. High fat feeding increases body lipid 76% and adipocyte size 65% in nontransgenic mice, but has no effect in transgenic mice. Thus, overexpression of GLUT4 selectively in adipocytes protects against a further increase in adiposity. Furthermore, by using a heterologous promoter, high level overexpression of GLUT4 can be maintained even under metabolic conditions where it is normally down-regulated in adipocytes. This overexpression results in markedly increased glucose transport at the cellular level, but adipose-specific GLUT4 overexpression does not prevent the decrease in glucose tolerance associated with high fat feeding.

Position independent expression and developmental regulation is directed by the beta myosin heavy chain gene's 5' upstream region in transgenic mice.

Transgenic mice generated with constructs containing 5.6 kb of the beta myosin heavy chain (MyHC) gene's 5' flanking region linked to the cat reporter gene express the transgene at high levels. In all 47 lines analyzed, tissue-specific accumulation of chloramphenicol acetyltransferase was found at levels proportional to the number of integrated transgene copies. Deletion constructs containing only 0.6 kb of 5' upstream region showed position effects in transgenic mice and did not demonstrate copy number dependence although transgene expression remained muscle-specific. The 5.6 kb 5' upstream region conferred appropriate developmental control of the transgene to the cardiac compartment and directs copy number dependent and position independent expression. Lines generated with a construct in which three proximal cis-acting elements were mutated showed reduced levels of transgene expression, but all maintained their position independence and copy number dependence, suggesting the presence of distinct regulatory mechanisms.

Chemical effects in transgenic mice bearing oncogenes expressed in mammary tissue.

Three transgenic mouse lines carrying v-Ha-ras (TG-SH), c-myc (TG-M) or c-neu (TG-NK) oncogenes under regulatory control of mouse mammary tumor virus (MMTV) long terminal repeat (LTR) sequences were evaluated for responses to two chemical carcinogens. p-Cresidine, a mutagenic urinary bladder carcinogen, increased the incidence of urinary bladder carcinomas in males and females in all three lines, and these tumors occurred at comparable incidences and grade in transgenic and non-transgenic mice. p-Cresidine did not affect the rates of mammary or salivary gland neoplasms in transgenic mice; these tumors did not occur in non-transgenic littermates. No other tumor types were observed in exposed or control animals. Reserpine, a non-mutagenic mammary gland carcinogen, was administered under the same protocol, but the high control rates of mammary gland adenocarcinomas in the TG-M and TG-NK strains made it difficult to detect any tumor-enhancing effect of reserpine. However, the incidences of multiple mammary gland tumors were significantly increased in dosed females from both lines. The incidence of mammary gland adenocarcinomas was significantly increased in TG-SH females receiving 5 p.p.m. reserpine. Reserpine did not induce any carcinogenic effects in non-transgenic mice. These results indicate that the transcriptional regulation of these three transgenes is a major determinant in the response to p-cresidine and reserpine. The use of transgenic models for the general detection of carcinogens may require lines in which appropriate genes are targeted for expression in many tissues, or lines in which critical genes have been inactivated.

Targeted replacement of the homeobox gene Hox-3.1 by the Escherichia coli lacZ in mouse chimeric embryos.

Through gene targeting based upon homologous recombination in embryonic stem cells, a chosen gene can be inactivated and eventually a strain of mutant mice created. We have devised a procedure to specifically replace a targeted gene by another gene. A murine homeobox gene was disrupted at high frequency in embryonic stem cells by its replacement with Escherichia coli lacZ. Injection of such stem cells into blastocysts yielded chimeric embryos in which beta-galactosidase activity was driven by the Hox-3.1 promoter. This technique will allow the visual assessment at the cellular level of gene inactivation effects in transgenic mice.