A missense mutation in the FZD7 gene is associated with dilution of the red areas of the coat in Montbéliarde cattle.

Recently, a new genetically autosomal recessive color phenotype emerged in the red pied bovine Montbéliarde breed. It is characterized by a dilution of the red areas of the coat and was denominated 'milca'. A genome-wide homozygosity scan of 106 cases followed by haplotype analysis revealed a candidate region within BTA2 between positions 89.95 and 91.63 Mb. Analysis of whole-genome sequence data generated from milca animals identified a strong candidate variant within the coding region of the Frizzled-7 gene (FZD7). This gene encodes for a G-protein coupled receptor for Wnt signaling proteins. The variant induces a glycine to alanine substitution in the second extracellular loop, p.(Gly414Ala). Cross-species amino acid alignments revealed that this glycine is conserved among orthologs and most paralogs, suggesting that it plays an important role in FZD function. In addition, genotyping data revealed that the mutant allele is restricted to the Montbéliarde breed, at a 3.7% frequency. All homozygous cows for the mutant allele exhibited the milca phenotype whereas all heterozygotes had no coat color defects. In conclusion, this study strongly suggests that, in cattle, a mutation of FZD7 alone is sufficient to cause a coat color phenotype without any strong other adverse effect.

Short communication: Mouse mammary tumor virus driven α-lactalbumin expression effects on lactation and fertility of transgenic mice.

α-Lactalbumin (Alac) is one of the major milk proteins. Its gene expression is restricted to epithelial cells of the lactating mammary gland. The Alac interaction with a uridine 5'-diphosphate-galactosyltransferase induces lactose synthesis, a major osmotic regulator of milk secretion. Other functions attributed to this protein include induction of apoptosis and anti-inflammatory activities. To assess if forced expression of this gene during early gestation or involution could affect mammary physiology, an Alac-encoding minigene was expressed in transgenic mice under the transcriptional regulation of the mouse mammary tumor virus promoter. The mammary expression did not interfere with gestation, resulted in a slight increase in milk yield as indirectly assessed by the 11% increased growth rate of the pups reared by transgenic females compared with that of those reared by control mice, and induced a slight delay in the early involution process, as demonstrated by histological analyses. The use of the mouse mammary tumor virus promoter resulted in Alac expression in several nonmammary tissues, such as the brain, the testis, the ovary, and the uterus. Although it did not affect male reproductive performances, it induced a female subfertile phenotype, characterized by embryonic implantation failure in the transgenic female reproductive tract.

Attempt to rescue sex-reversal by transgenic expression of the PISRT1 gene in XX PIS-/- goats.

The Polled Intersex Syndrome (PIS mutation) in goats leads to an absence of horn and to an early sex-reversal of the XX gonads. This mutation is a deletion of an 11.7-kb DNA fragment showing a tissue-specific regulatory activity. Indeed, in XX PIS(-/-) gonads the deletion of PIS leads to the transcriptional extinction of at least 3 neighboring genes, FOXL2, PFOXic and PISRT1. Among them, only FOXL2 is a 'classical' gene, encoding a highly conserved transcription factor. On the other hand, knock-out of Foxl2 in mice results in an early blocking of follicle formation without sex-reversal. This phenotype discrepancy leads to two hypotheses, either FOXL2 is responsible for XX sex-reversal in goat assuming distinct functions of its protein during ovarian differentiation in different mammals, or other PIS-regulated genes are involved. To assess the second possibility, PISRT1 expression was constitutively restored in XX PIS(-/-) gonads. Six transgenic fetuses were obtained by nuclear transfer and studied at 2 developmental stages, 41 and 46 days post-reconstruction. The gonads of these fetuses appear phenotypically identical to those of cloned non-transgenic controls. Conclusively, this result argues for FOXL2 being responsible for the PIS gonad-associated phenotype. Its invalidation in goat will help to better understand this complex syndrome.

Goat PRND expression pattern suggests its involvement in early sex differentiation.

Expression of the goat prion protein gene locus was assessed by reverse transcriptase-polymerase chain reaction on testes and ovaries at various developmental stages. A weak and stochastic expression of the PRNP and PRNT genes was observed. For PRNT, it is consistent with the detected deletions of two single nucleotides within its open reading frame in ruminant genes. PRND was expressed in both tissues at all stages. Whereas its expression is constant in the ovaries, it increases in testes between 36 and 46 days postcoitum (dpc) and remains high thereafter. In testes, Doppel was found in the nucleus of germinal cells and in the cytoplasm of Leydig cells at 44 dpc. It was detected in the cytoplasm of Leydig cells and of some Sertoli and germinal cells at 62 dpc. In the ovaries, it was observed in the nucleus of germinal cells at 44 dpc and mainly in their cytoplasm at 62 dpc. This expression pattern was shown to parallel that of C-kit and suggests Doppel involvement in early testis differentiation.

Unexpected high testis-specific transcriptional activity of the cyclin T1 promoter in transgenic mice.

The ubiquitously expressed cyclin T1 gene encodes for a protein involved in human immunodeficiency virus type 1 (HIV-1) transcription activation. The goat gene was recently shown to share an expression pattern similar to that of its endogenous counterpart when incorporated into mice using a BAC insert. To assess if its promoter could target ubiquitous expression of the bovine Prnp in transgenic mice, two constructs carrying either 1 or 30 kb of cyclin T1 5'-flanking sequences were built and microinjected. Both constructs resulted in the unexpected high male germ cell-specific expression of the prion protein. These data re-question the suspected location of the cyclin T1 gene regulatory elements.

Efficient and specific down-regulation of prion protein expression by RNAi.

Prion diseases are fatal neurodegenerative disorders associated with an abnormal isoform of the PrPc host-encoded protein. Invalidation of the Prnp gene, that encodes PrPc, led to transgenic mice that are viable, apparently healthy, and resistant to challenge by the infectious agent. These results indicated that a down-regulation of the Prnp gene expression is a potential therapeutic approach. In the present report, we demonstrate that RNAi targeted towards the Prnp mRNA can efficiently and highly specifically reduce the level of PrPc in transfected cells. It, thus, indicates that RNAi is an attractive therapeutic approach to fight against prion diseases.

Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine prp.

The susceptibility of sheep to scrapie is known to involve, as a major determinant, the nature of the prion protein (PrP) allele, with the VRQ allele conferring the highest susceptibility to the disease. Transgenic mice expressing in their brains three different ovine PrP(VRQ)-encoding transgenes under an endogenous PrP-deficient genetic background were established. Nine transgenic (tgOv) lines were selected and challenged with two scrapie field isolates derived from VRQ-homozygous affected sheep. All inoculated mice developed neurological signs associated with a transmissible spongiform encephalopathy (TSE) disease and accumulated a protease-resistant form of PrP (PrPres) in their brains. The incubation duration appeared to be inversely related to the PrP steady-state level in the brain, irrespective of the transgene construct. The survival time for animals from the line expressing the highest level of PrP was reduced by at least 1 year compared to those of two groups of conventional mice. With one isolate, the duration of incubation was as short as 2 months, which is comparable to that observed for the rodent TSE models with the briefest survival times. No survival time reduction was observed upon subpassaging of either isolate, suggesting no need for adaptation of the agent to its new host. Overexpression of the transgene was found not to be required for transmission to be accelerated compared to that observed with wild-type mice. Conversely, transgenic mice overexpressing murine PrP were found to be less susceptible than tgOv lines expressing ovine PrP at physiological levels. These data argue that ovine PrP(VRQ) provided a better substrate for sheep prion replication than did mouse PrP. Altogether, these tgOv mice could be an improved model for experimental studies on natural sheep scrapie.

Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein.

Transmissible spongiform encephalopathies, or prion diseases, are fatal degenerative disorders of the central nervous system that affect humans and animals. Prions are nonconventional infectious agents whose replication depends on the host prion protein (PrP). Transmission of prions to cultured cells has proved to be a particularly difficult task, and with a few exceptions, their experimental propagation relies on inoculation to laboratory animals. Here, we report on the development of a permanent cell line supporting propagation of natural sheep scrapie. This model was obtained by stable expression of a tetracycline-regulatable ovine PrP gene in a rabbit epithelial cell line. After exposure to scrapie agent, cultures were repeatedly found to accumulate high levels of abnormal PrP (PrPres). Cell extracts induced a scrapie-like disease in transgenic mice overexpressing ovine PrP. These cultures remained healthy and stably infected upon subpassaging. Such data show that (i) cultivated cells from a nonneuronal origin can efficiently replicate prions; and (ii) species barrier can be crossed ex vivo through the expression of a relevant PrP gene. This approach led to the ex vivo propagation of a natural transmissible spongiform encephalopathy agent (i.e., without previous experimental adaptation to rodents) and might be applied to human or bovine prions.

Distal element(s) is(are) required for position-independent expression of the goat alpha-lactalbumin gene in transgenic mice. Potential relationship with the location of the cyclin T1 locus.

We recently reported the site-independent and copy-number-related expression in mice of a goat alpha-lactalbumin gene with 150 kb and 10 kb of 5'- and 3'-flanking sequences, respectively. In the present study, we observed that the resection of the 5'-flanking region, leaving only 70 kb, resulted in a site-dependent expression of this milk protein-encoding transgene. This suggests that important cis-regulatory elements are located within the distal-deleted sequence. Within this region, we localised the promoter of the cyclin T1 gene, an ubiquitously expressed gene. So far, no other gene has been located between these two loci. Since these two genes are differentially expressed, our data suggest the potential location of an insulator within the deleted region that allows the two genes to be independently regulated.

Introduction of a proximal Stat5 site in the murine alpha-lactalbumin promoter induces prolactin dependency in vitro and improves expression frequency in vivo.

In order to establish a possible correlation between in vitro prolactin induction and the transcriptional activity of mammary gene promoters in transgenic mice, a functional Stat5-binding site was created by means of site-directed mutagenesis at position -70 on a 560 bp murine alpha-lactalbumin promotor linked to a CAT reporter gene. Surprisingly, the wild-type promoter was constitutively active in vitro and could not be induced by prolactin. Introducing the proximal Stat5 site abolished this constitutive activity and resulted in prolactin dependence in both CHO-K1- and HC11-transfected cells. In transgenic mice, both the frequency of lines expressing the transgene and the prevalence of mid to late pregnancy expression were increased.

Use of doxycycline-controlled gene expression to reversibly alter milk-protein composition in transgenic mice.

A reverse tetracycline transactivator-encoding cDNA under the control of the mammary specific beta-lactoglobulin promoter was linked to a bovine alpha-lactalbumin transcription unit driven by a reverse tetracycline-controlled transactivator/doxycycline-inducible human cytomegalovirus promoter. The construct was microinjected into eggs from alpha-lactalbumin-deficient mice. These mice produce a highly viscous lactose-free milk and have a shortened lactation period. Mice from three out of the nine transgenic lines investigated expressed reverse tetracycline-controlled transactivator mRNA in their lactating mammary glands at levels detectable by Northern analysis. Following doxycycline addition to the drinking water, lactation was fully restored in animals from the three lines. Doxycycline removal resulted in a reversal of phenotype. The observed mammary-specific and high expression of the doxycycline inducible reporter gene (up to 5.2 mg of recombinant alpha-lactalbumin.mL-1 of milk, i.e. up to 13-fold induction) opens up exciting prospects to use the tetracycline system to study the development and functioning of the mammary gland, and to control the production level of active pharmaceutical proteins in the milk of transgenic animals.

Position-independent and copy-number-related expression of a goat bacterial artificial chromosome alpha-lactalbumin gene in transgenic mice.

A bacterial artificial chromosome goat insert comprising the alpha-lactalbumin-encoding transcription unit with approximately 150 and 10 kb of 5'- and 3'-flanking sequences, respectively, was micro-injected into mouse eggs. In six out of seven transgenic lines, the level of mammary tissue- and stage-specific expression was position-independent and copy-number-dependent. The exogenous alpha-lactalbumin yield, about 0.8 mg/ml of milk per copy, compared favourably with the alpha-lactalbumin content of mouse and goat milks, about 0.8 and >1 mg/ml, respectively. This suggests that the insert contains most if not all of the cis-acting elements involved in the full and specific expression of the goat alpha-lactalbumin gene and opens up opportunities to use this vector to target expression of foreign genes in the lactating mammary gland of transgenic animals. The transgene was silent in the seventh line for an unknown reason.

Production of low-lactose milk by ectopic expression of intestinal lactase in the mouse mammary gland.

We have investigated, in mice, an in vivo method for producing low-lactose milk, based on the creation of transgenic animals carrying a hybrid gene in which the intestinal lactase-phlorizin hydrolase cDNA was placed under the control of the mammary-specific alpha-lactalbumin promoter. Transgenic females expressed lactase protein and activity during lactation at the apical side of mammary alveolar cells. Active lactase was also secreted into milk, anchored in the outer membrane of fat globules. Lactase synthesis in the mammary gland caused a significant decrease in milk lactose (50-85%) without obvious changes in fat and protein concentrations. Sucklings nourished with low-lactose milk developed normally. Hence, these data validate the use of transgenic animals expressing lactase in the mammary gland to produce low-lactose milk in vivo, and they demonstrate that the secretion of an intestinal digestive enzyme into milk can selectively modify its composition.

Sequence of murine CDC10 cDNA, gene organization and expression analysis.

The sequence of the 2391 bp murine CDC10 cDNA is reported. The gene transcription unit, composed of 13 exons, encodes a potential 417/446 amino acid GTP-binding protein, highly similar to human CDC10. The ubiquitous expression of the gene, as judged by Northern analysis, is consistent with its putative promoter structure.

Lactase is unchanged in suckling mice fed with lactose-free milk.

At weaning, mammals switch from milk to complex adult food, and change from a lactose-rich to a lactose-free diet. At the same time, the small intestine matures resulting in changes in lactase expression and the onset of sucrase. The aim of this study was to analyze the effect of premature and specific depletion of lactose on maturation of the small intestine and on lactase expression in suckling mice. For this purpose, from postnatal days 10 to 16, suckling mice were fed by transgenic alpha-lactalbumin-deficient females that produce lactose-free milk. Pups fed with lactose-free milk had a lower body weight than controls fed by wildtype females. They also displayed hypotrophy of intestinal muscle layers, but no obvious alterations in the morphology of the intestinal epithelium. The level of lactase activity as well as the longitudinal distribution of corresponding mRNA were unchanged compared to suckling animals nourished with normal lactose-containing milk. Finally, there was no premature onset of sucrase expression. We conclude that feeding suckling mice for six days with lactose-free milk does not provoke any premature maturation of the small intestine. Thus, decreasing lactose intake is not a major cause for the modifications of lactase expression which occur at weaning.

Modification and repression of genes expressed in the mammary gland using gene targeting and other technologies.

Transgenic experiments using oocyte micro-injection methodology are often performed in order to target expression of a foreign gene in a specific tissue or, to a lesser extent, to study the regulation of gene expression. However, the isolation of embryonic stem cells in mice and the development of antisense and ribozyme technologies have allowed more subtle alterations of endogenous gene expression to be achieved. The mammary gland is one of the few organs able to undergo several cycles of development, differentiation and apoptosis through complex multihormonal regulation during adult life. It is thus an attractive model to assess the in vivo function of some genes potentially involved in these mechanisms, either by silencing them or by partially repressing their expression. Furthermore, such alterations of gene expression have also been performed for more applied objectives such as the modification of milk composition for nutritional and technological purposes. This review will describe the experimental procedures used toward these aims and the results already obtained in this field. Some potential new targets will be suggested.

Rescue of an MMTV transgene by co-integration reveals novel locus control properties of the ovine beta-lactoglobulin gene that confer locus commitment to heterogeneous tissues.

In an attempt to enhance the frequency and level of expression of a poor-performing MMTV-driven transgene, we co-integrated this construct with the ovine beta-lactoglobulin (BLG) gene in transgenic mice. Seven lines of transgenic mice possessing co-integrated BLG and MMTV-RZ5 transgenes were compared with 12 lines of mice that possessed only the MMTV-RZ5 construct. Co-integration enhanced the frequency of expression in the mammary gland from two out of 12 lines for the MMTV-RZ5 transgene alone, to five out of seven when co-integrated with BLG. Surprisingly, co-integration also resulted in co-expression of the two transgenes in the salivary gland, lung and spleen in addition to the mammary gland. Furthermore, both transgenes were expressed in virgin animals, and throughout pregnancy and lactation, suggesting that the developmental regulation of the locus follows that of the MMTV-promoter. These findings represent a novel locus control property of the ovine BLG gene that confers commitment of the locus to the mammary gland, but also to a range of heterogeneous tissues possibly defined by the second promoter at the locus.

Developmental regulation of murine integrin beta 1 subunit- and Hsc73-encoding genes in mammary gland: sequence of a new mouse Hsc73 cDNA.

A partial integrin beta 1 subunit-encoding cDNA (Itg beta 1) and a new heat-shock protein 70-like-encoding cDNA (Hsc73) homologous to rat Hsc73 were cloned by differential display and RT-PCR from mouse mammary gland. Their developmental regulation during pregnancy, lactation and involution is reported. The Itg beta 1 mRNA content was stable in the first half of gestation, decreased to a minimum during lactation and increased markedly in early involution. Hsc73 gene expression was high in the first half of gestation and decreased to a minimum during lactation. The possible significance of the two observed patterns of expression is discussed.

Efficient and specific ribozyme-mediated reduction of bovine alpha-lactalbumin expression in double transgenic mice.

Transgenic mice carrying a bovine alpha-lactalbumin (alpha-lac) specific ribozyme gene under the transcriptional control of the mouse mammary tumor virus long terminal repeat were generated and cross-bred with animals that highly express a bovine alpha-lac transgene (0.4 mg of alpha-lac/ml(-1) of milk). The ribozyme contains the hammerhead catalytic domain, flanked by 12-nt sequences complementary to the 3' untranslated region of bovine alpha-lac transcript. High-level expression of the ribozyme gene was detected by Northern blot analysis in the mammary gland of 7-8 day lactating transgenic mice, from 3 of 12 lines analyzed. Heterozygous expression of the ribozyme resulted in a reduction in the levels of the target mRNA to 78, 58, and 50% of that observed in the nonribozyme transgenic littermate controls for three independent lines. The ribozyme-mediated reduction in the levels of the bovine protein paralleled that observed for the mRNA, and was positively correlated with the level of expression of the ribozyme. In nonribozyme expressing transgenic mice, the level of bovine alpha-lac mRNA and protein was not affected. The specificity of this activity is demonstrated by the absence of a reduction in the levels of the endogenous murine alpha-lac mRNA or protein. These results demonstrate the feasibility of ribozyme-mediated down-regulation of highly-expressed transcripts in transgenic animals.