Expression of the chicken transferrin gene in transgenic mice

The chicken transferrin gene was microinjected into the male pronucleus of fertilized mouse eggs, and the eggs were then implanted into foster mothers. Approximately 15%-30% of the offspring from the injected eggs carried chicken DNA sequences; restriction mapping indicated that multiple copies of the chicken gene had integrated into the genome in a tandem arrangement in most of the mice. Six of the seven mice studied expressed the chicken gene, and in five mice there was a 5 to 10 fold preferential expression of chicken transferrin mRNA in liver compared to that in other tissues. Chicken transferrin was secreted into the serum of five of the mice, where it reached steady state concentrations up to 67 micrograms/ml. Offspring from transgenic parents also expressed the chicken gene; in some cases the expression in offspring was very similar to the parent, but in one line expression in offspring had increased 2 to 4 fold.     

Developmental regulation of the human zeta globin gene in transgenic mice.

We have characterized the expression of the human zeta (zeta) gene, which encodes an embryonic alpha-like globin, in transgenic mice. We find that a 777 base pair fragment spanning erythroid specific hypersensitive site II (HSII) from the distal 5. region of the human beta globin gene cluster potentiates expression of the zeta globin gene. In the absence of the HSII fragment, no zeta expression is observed. Expression of the human zeta gene in mice parallels expression of a murine embryonic alpha-like globin gene (x). Thus, expression of the human zeta gene in mice requires linkage to an erythroid-specific enhancer sequence, but the presence of the enhancer does not affect the developmental regulation of the transgene. Our results indicate that the factors involved in switching from embryonic to adult alpha globin gene expression during development are evolutionarily conserved, and suggest that the transgenic mouse is an in vivo system in which the requirements for the developmental switch in alpha globin gene expression can be analyzed in detail.     

Expression of a human chimeric transferrin gene in senescent transgenic mice reflects the decrease of transferrin levels in aging humans.

Transgenic mice provide a means to study human gene expression in vivo throughout the aging process. A DNA sequence containing 668 bp of the 5' regulatory region of the human transferrin gene was fused to the bacterial reporter gene chloramphenicol acetyl transferase (TF-CAT) and introduced into the mouse genome. Expression of the human chimeric transferrin gene was similar to the tissue patterns of mouse and human transferrin. In aging transgenic mice, expression of the human chimeric transferrin gene was found to diminish 40% in livers between 18 and 26 months of age. Transferrin levels and serum iron levels in aging humans also diminish, as observed from measurements of total iron binding capacity and percent iron saturation in sera from 701 individuals ranging from 0 to 99 years of age. In contrast, in transgenic mice and nontransgenic mice, the mouse endogenous plasma transferrin and endogenous Tf mRNA increase significantly during aging. Neither the decrease of human TF-CAT nor the increase of mouse transferrin during aging appears to be part of a typical inflammatory reaction. Although the 5' regions of the human transferrin and mouse transferrin genes are homologous, sequence diversities exist which could account for the different responses to inflammation and aging observed.     

Differential regulation of the N-myc gene in transfected cells and transgenic mice.

The N-myc gene is expressed specifically in the early developmental stages of numerous cell lineages. To assay for sequences that could potentially regulate N-myc expression, we transfected constructs that contained murine N-myc genomic sequences linked to a reporter gene and genomic clones that contained the complete human or murine N-myc genes into cell lines that either express or do not express the endogenous N-myc gene. Following either transient or stable transfection, the introduced N-myc sequences were expressed regardless of the expression status of the endogenous gene. In contrast, when the clones containing the complete human N-myc gene were introduced into the germline of transgenic mice, expression in some transgenic lines paralleled the tissue- and stage-specific expression of the endogenous murine gene. These findings demonstrate differences in the regulation of N-myc genes in recipient cells following in vitro versus in vivo introduction, suggesting that early developmental events may play a role in the regulation of N-myc expression.     

Expression and regulation of the rabbit uteroglobin gene in transgenic mice

The rabbit uteroglobin (UG) gene, with varying lengths of 5' flanking sequence, was introduced into the mouse genome to investigate the DNA sequences required for tissue-specific expression and regulation by steroid hormones. The pattern of expression and steroid hormone regulation of the transgene was compared to the expression and regulation of the endogenous mouse UG-like gene. In the rabbit, UG is induced in the uterus by progesterone and is expressed constitutively in the lungs, where it is weakly regulated by glucocorticoids. Genomic DNA fragments containing the complete UG-coding sequence with 4.0 (UG4.0), 3.0 (UG3.0), 2.3 (UG2.3), or 0.6 (UG0.6) kilobases of 5' flanking sequence were used to establish lines of transgenic mice. Expression of UG mRNA was observed in the lungs of UG4.0 (2/4 lines), UG3.0 (4/4 lines), UG2.3 (1/2 lines), and UG0.6 (4/4 lines) mice. Uterine expression was observed in UG3.0 (3/4 lines), UG2.3 (1/2 lines), and UG0.6 (2/4 lines). In the lungs of UG3.0 and UG2.3 mice, RNA expression was stimulated by treatment with dexamethasone. In the one line of UG3.0 mice examined, UG was regulated by ovarian steroids in the uterus. The endogenous mouse UG-like gene showed the major site of expression to be in the lung. Unlike the transgene, the endogenous gene was more strongly stimulated by glucocorticoids. Thus, we conclude that the cis elements needed for pulmonary expression of UG are contained within the UG2.3 fragment used to generate transgenic mice, but that other elements are required for full glucocorticoid regulation. Also, the transgene did not show the full uterine expression observed in the rabbit, but regulation by the ovarian steroids was observed.     

Differential regulation of rat beta-casein-chloramphenicol acetyltransferase fusion gene expression in transgenic mice.

Previous studies in our laboratory have demonstrated the mammary-specific expression of the entire rat beta-casein gene with 3.5 kilobases (kb) of 5' and 3.0 kb of 3' DNA in transgenic mice (Lee et al., Nucleic Acids Res. 16:1027-1041, 1988). In an attempt to localize sequences that dictate this specificity, lines of transgenic mice carrying two different rat beta-casein promoter-bacterial chloramphenicol acetyltransferase (cat) fusion genes have been established. Twenty and eight lines of transgenic mice carrying two fusion genes containing either 2.3 or 0.5 kb, respectively, of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A were identified, most of which transmitted the transgenes to their offspring in a Mendelian pattern. CAT activity was detected predominantly in the lactating mammary gland of female transgenic mice but not in the male mammary fat pad. A several-hundred-fold variation in the level of cat expression was observed in the mammary gland of different lines of mice, presumably due to the site of integration of the transgenes. CAT activity was increased in the mammary gland during development from virgin to midpregnancy and lactation. Unexpectedly, the casein-cat transgenes were also expressed in the thymus of different lines of both male and female mice, in some cases at levels equivalent to those observed in the mammary gland, and in contrast to the mammary gland, CAT activity was decreased during pregnancy and lactation in the thymus. Thus, 0.5 kb of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A are sufficient to target bacterial cat gene expression to the mammary gland of lactating mice.     

Developmental regulation for collagen II gene expression in transgenic mice

In order to evaluate the involvement of the type II collagen regulatory sequences in development, we have injected a construct containing a toxin gene under the control of the rat type II collagen promoter and enhancer. The construct, pDAS10-DTA, contained the diphtheria toxin A chain gene under the control of type II collagen sequences which had been used previously to target cartilagenous tissues in transgenics. Inspection of developing fetuses at various stages of gestation revealed a high number of aborted implants as well as abnormally developing fetuses. These abnormal fetuses were of small size, had shortened and underdeveloped limbs, cleft palates, and generally resembled a phenotype similar to chondrodystrophic mice. Histological comparisons of normal and abnormal fetuses indicated a reduced amount of extracellular matrix surrounding chondrocytes, and a disorganized appearance of the tissue. These results suggest that the expression of the toxin has occurred in chondrocytes and altered the survival and development of the transgenic mice. These results also indicate that the promoter and enhancer sequences contained in the transgene controlled the developmental expression of the type II collagen gene expression.     

Sexual dimorphism and growth hormone regulation of a hybrid gene in transgenic mice.

The sexually dimorphic expression of the urinary protein genes of mice (Mup genes) in the liver is mediated by the different male and female temporal patterns of circulating GH. Normal females were induced to male levels when GH was administered by injection to mimic the male GH pattern, showing that expression at the male level does not require a male sex steroid status in addition to intermittent GH. Two Mup-alpha 2u-globulin hybrid transgenes with different Mup gene promoters showed sexually dimorphic expression, and their expression in females increased to male levels upon testosterone treatment. GH-deficient (lit/lit) mice did not express these transgenes, and GH-deficient females did not respond to testosterone treatment, showing that GH was required for induction. Both normal and GH-deficient females were induced to male levels when GH was administered by injection. This is the first report of a transgene responsive to GH. A transgene consisting of a Mup promoter fused to a Herpes simplex virus thymidine kinase reporter sequence also showed sexual dimorphism, although to a lesser degree. It was expressed at the same level in normal females and GH-deficient mice of both sexes and was induced when GH-deficient mice were treated with GH. We propose that this transgene has a basal constitutive expression, possibly due to the absence of any rodent DNA downstream of the promoter. Since expression of the transgene was significantly induced by GH, the GH response is due at least in part to sequences in the promoter region.     

The developmental regulation of the human zeta-globin gene in transgenic mice employing beta-galactosidase as a reporter gene.

We have investigated the developmental and tissue specific expression of the human embryonic zeta-globin gene in transgenic mice. A construct containing 550 bp of zeta-globin 5' flanking region, fused to a beta-galactosidase (lacZ) reporter gene and linked to the locus control region (LCR)-like alpha positive regulatory element (alpha PRE) was employed for the production of transgenic mice. Firstly, we compared the number of live born transgenic mice containing this construct to the number of live born transgenic mice containing the entire zeta-globin gene linked to the alpha PRE or the beta LCR. Data showed that 12% of mice generated from eggs injected with zeta-promoter/lacZ/alpha PRE DNA were transgenic compared to only 2% of mice generated from eggs injected with the entire zeta-globin gene linked to the alpha PRE or the beta LCR. The reduced number of live born transgenic mice containing the latter constructs suggests that death of transgenic embryos, possibly due to thalassaemia, may be occurring. X-gal staining of whole embryos containing the lacZ gene revealed that zeta-globin promoter activity was most pronounced at 8.5-9.5 days of development and was restricted to erythroid cells. By 15 days of development, no zeta-globin promoter activity was detected. These results suggest that the alpha PRE can direct high level expression from the zeta-globin promoter and that sequences required for the correct tissue and developmental specific expression of the human zeta-globin gene are present within 550 bp's of 5' flanking region. Sequences within the body of the zeta-globin gene or 3' of the cap site do not appear to be necessary for correct zeta-globin developmental regulation.     

Differential regulation of the rat phosphoenolpyruvate carboxykinase gene expression in several tissues of transgenic mice.

The selective expression of a unique copy gene in several mammalian tissues has been approached by studying the regulatory sequences needed to control expression of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene in transgenic mice. A transgene containing the entire PEPCK gene, including 2.2 kb of the 5'-flanking region and 0.5 kb of the 3'-flanking region, exhibits tissue-specific expression in the liver, kidney, and adipose tissue, as well as the hormonal and developmental regulation inherent to endogenous gene expression. Deletions of the 5'-flanking region of the gene have shown the need for sequences downstream of position -540 of the PEPCK gene for expression in the liver and sequences downstream of position -362 for expression in the kidney. Additional sequences upstream of position -540 (up to -2200) are required for expression in adipose tissue. In addition, the region containing the glucocorticoid-responsive elements of the gene used by the kidney was identified. This same sequence was found to be needed specifically for developmental regulation of gene expression in the kidney and, together with upstream sequences, in the intestine. The apparently distinct sequence requirements in the various tissues indicate that the tissues use different mechanisms for expression of the same gene.     

Tissue-specific and hormonal regulation of the gene for rat prostatic steroid-binding protein in transgenic mice.

We investigated the tissue-specific and hormonal regulation of the gene for rat prostatic steroid-binding protein by introducing the C3(1) gene with 4-kilobase (kb) upstream and 2-kb downstream flanking sequences into transgenic mice. There was selective expression in the ventral prostate that was stimulated by testosterone, which indicated that the gene together with 6-kb flanking DNA contains the information required for prostate-specific and testosterone-regulated expression.     

Human transferrin. Expression and iron modulation of chimeric genes in transgenic mice

Transferrin (TF) is a plasma protein that transports and is regulated by iron. The aim of this study was to characterize human TF gene sequences that respond in vivo to cellular signals affecting expression in various tissues and to iron administration. Chimeric genes were constructed containing 152, 622, and 1152 base pairs (bp) of the human TF5'-flanking region with the coding region of a reporter gene, CAT (chloramphenicol acetyltransferase), and introduced into the germ line of mice. Transgenes containing TF 5'-flanking sequences to -152 bp were expressed poorly in all tissues examined. In contrast, transgenes containing TF sequences to -622 or -1152 bp were expressed at high levels in brain and liver, greater than or equal to 1000-fold higher than tissues such as heart and testes. Liver and brain are major sites of endogenous TF mRNA synthesis, but liver mRNA levels are 10-fold higher than brain. A significant diminution of CAT enzymatic activity in liver accompanied iron administration in both TF(0.67) and TF(1.2)CAT transgenic mice, mimicking the decrease of transferrin in humans following iron overload. Levels of endogenous plasma transferrin also decreased in iron-treated transgenic mice. Transgenic mouse lines carrying human TF chimeric genes will be useful models for analyzing the regulation of human transferrin by iron and for determining the molecular basis of transferrin regulation throughout mammalian development into the aging process.     

Expression and regulation of a proenkephalin beta-galactosidase fusion gene in the reproductive system of transgenic mice.

A fusion gene containing 3 kilobases of human proenkephalin 5'-flanking sequences and 1 kilobase of human proenkephalin 3'-flanking sequence and the easily visualized histochemical marker, Escherichia coli beta-galactosidase, was used to study the function of cis-regulatory elements within the human proenkephalin gene in transgenic mice. Here data are presented on expression and regulation of this fusion gene in the reproductive system of three independent lines of transgenic mice. Within the male reproductive system, the fusion gene is expressed in the proximal epididymis and in developing germinal cells but not in mature or elongating spermatids. In the female reproductive system, the transgene was expressed at low basal levels, but expression was dramatically stimulated in the ovary and oviduct by hormonal stimulation and pregnancy; additionally, expression was induced at the uteroplacental junction in pregnant mice. Taken together these observations suggest that critical sequences for expression and regulation of the proenkephalin gene within the reproductive system are contained within sequences of the construct.