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L M Houdebine

Publications and source records attributed to L M Houdebine.

At least 73 records · Page 4Linked to original sources
PubMed1992

A distal region enhances the prolactin induced promoter activity of the rabbit alpha s1-casein gene.

Casein gene expression is induced in the rabbit mammary gland by prolactin (PRL). alpha s1-casein is the major casein secreted into milk. In order to define the position of the DNA sequences involved in the control of rabbit alpha s1-casein gene regulation by PRL, chimeric genes were constructed between upstream regions of the rabbit alpha s1-casein gene and the chloramphenicol acetyl transferase (CAT) reporter gene. A series of 5'-deleted fusion genes was obtained by nuclease digestion of the alpha s1-casein gene upstream region. These gene constructs were transfected into rabbit primary mammary cells, or cotransfected in CHO cells with the plasmid coding for the rabbit mammary receptor (PRL-R). A regulatory region has been located between nt -3768 and -3155. This region enhances the prolactin induced promoter activity of the alpha s1-casein gene. It might possess or cooperate with prolactin responsive elements located further downstream in the alpha s1-casein gene.

Animals↗
PubMed1991

Transgenesis in fish.

Gene transfer into fish embryo is being performed in several species (trout, salmon, carps, tilapia, medaka, goldfish, zebrafish, loach, catfish, etc.). In most cases, pronuclei are not visible and microinjection must be done into the cytoplasm of early embryos. Several million copies of the gene are generally injected. In medaka, transgenesis was attempted by injection of the foreign gene into the nucleus of oocyte. Several reports indicate that the injected DNA was rapidly replicated in the early phase of embryo development, regardless of the origin and the sequence of the foreign DNA. The survival of the injected embryos was reasonably good and a large number reached maturity. The proportion of transgenic animals ranged from 1 to 50% or more, according to species and to experimentators. The reasons for this discrepancy have not been elucidated. In all species, the transgenic animals were mosaic. The copy number of the foreign DNA was different in the various tissues of an animal and a proportion lower than 50% of F1 offsprings received the gene from their parents. This suggests that the foreign DNA was integrated into the fish genome at the two cells stage or later. An examination of the integrated DNA in different cell types of an animal revealed that integration occurred mainly during early development. The transgene was found essentially unrearranged in the fish genome of the founders and offsprings. The transgenes were therefore stably transmitted to progeny in a Mendelian fashion. Southern blot analysis revealed the presence of possible junction fragments and also of minor bands which may result from a rearrangement of the injected DNA. In all species, the integrated DNA appeared mainly as random end-to-end concatemers. In adult trout blood cells, a small proportion of the foreign DNA was maintained in the form of non-integrated concatemers, as judged by the existence of end fragments. The transgenes were generally only poorly expressed. The majority of the injected gene constructs contained essentially mammalian or higher vertebrates sequences. The comparison of the expression efficiency of these constructs in transfected fish and mammalian cells indicates that some of the mammalian DNA sequences are most efficiently understood by the fish cell machinery. Chloramphenicol acetyl transferase gene under the control of promoters from Rous sarcoma virus, and human cytomegalovirus, was expressed in several tissues of transgenic fish. Chicken delta-crystallin gene was expressed in several tissues of transgenic fish.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Structure of the gene encoding rabbit beta-casein.

The entire rabbit beta-casein-encoding gene and 400 bp upstream were sequenced. Eight introns, located essentially at a position similar to the corresponding gene in other species, were found. Strong homology with several casein-encoding genes from rabbit and from other species was observed in the upstream region of the gene. Repeated sequences of unknown function were also located within introns.

Amino Acid Sequence↗
PubMed1991

Expression of active recombinant human alpha 1-antitrypsin in transgenic rabbits.

A DNA construct containing the human alpha 1-antitrypsin gene including 1.5 and 4 kb of 5' and 3' flanking sequences, was microinjected into the pronucleus of rabbit embryos. The recombinant human protein was (a) expressed in the blood circulation of F0 and F1 transgenic rabbits at an average concentration of 1 mg ml-1, (b) shown to be fully active and (c) shown to be separable from its rabbit counterpart. Transgenic rabbits might represent a novel source of human proteins of therapeutic interest.

Animals↗
PubMed1991

Hormone responsive elements within the upstream sequences of the rabbit whey acidic protein (WAP) gene direct chloramphenicol acetyl transferase (CAT) reporter gene expression in transfected rabbit mammary cells.

Whey acidic protein gene transcription is induced in the mammary gland under the influence of lactogenic hormones: prolactin, insulin and cortisol. The rabbit WAP gene has already been isolated and sequenced in a previous work. In the present study, we have evaluated the role of the 5' flanking region of the rabbit WAP gene in the transcriptional regulation of the WAP gene by using a reporter CAT gene. Chimeric genes containing the upstream region of the WAP gene have been linked to the bacterial CAT gene and transfected into rabbit primary mammary cells. The results reported here show that two regions carrying important regulatory elements of the rabbit WAP gene are located between -6300 and -3000 bp, and between -3000 and -1800 bp upstream from the WAP transcription start point, respectively. The contribute to the high level of expression of the rabbit WAP gene in the mammary cell.

Animals↗
PubMed1991

Cortisol induces rapid accumulation of whey acid protein mRNA but not of asl and b-casein mRNA in rabbit mammary explants.

Mammary explants from rabbit were cultured in the presence of various combinations of insulin, cortisol and prolactin. The concentration of whey acidic protein (WAP) asl-casein and b-casein mRNA was measured using specific cDNA probes. Medium alone was added to the explants for one day. Prolactin with and without cortisol was then added to the medium. Prolactin alone induced rapidly asl-and b-casein gene but not WAP gene. When cortisol was added with prolactin, the asl- and b-casein genes were induced at the same rate as in the absence of the steroid. In contrast, the WAP mRNA was then rapidly accumulated. This induction process was not altered by cycloheximide for two hours and it was blocked at a later stage. In a second experiment, insulin and prolactin were first added for 24 hours in the culture medium. Cortisol was then added and the concentration of the three mRNA was measured. Cortisol did not significantly modify the level of asl- and b-casein mRNA. On the contrary, the WAP mRNA was rapidly accumulated. These data indicate that the well-established amplificatory effect of glucocorticoids on casein gene expression is a slow process whereas their effect on the WAP gene is rapid. This suggests that glucocorticoids induce casein gene expression through an indirect cellular mechanism not involving a glucocorticoid receptor element in casein gene promoters and that WAP gene is more classically stimulated through the direct binding of the steroid receptor to a glucocorticoid receptor element located in its promoter.

Actins↗
PubMed1991

Rabbit whey acidic protein concentration in milk, serum, mammary gland extract, and culture medium.

Rabbit whey acidic protein has been purified from whey using an AcA54 column. The purified whey acidic protein had an amino acid composition in agreement with the previously defined cDNA sequence. An antibody against whey acidic protein was raised in guinea pig. This antibody did not crossreact with mouse or cow milk or with rabbit alpha s1-casein and beta-casein. Whey acidic protein concentration was measured in rabbit milk using the antibody with a radioimmunoassay. The concentration of whey acidic protein in rabbit milk was 15 mg/ml, whereas the concentrations of alpha s1-casein and beta-casein were 16 and 45 mg/ml, respectively. The concentration of the three proteins was also evaluated in culture medium of rabbit primary mammary cells. The three proteins were induced by prolactin alone. Glucocorticoids amplified the prolactin effect on whey acidic protein more intensively than on caseins. The three proteins were present in mammary extract from virgin rabbit. The concentration of these proteins was lower at d 8 and 14 of pregnancy, and it was very high at d 25 of pregnancy. Whey acidic protein was undetectable in blood of virgin, weaned, and midpregnant females and of males. Whey acidic protein was present in blood of lactating rabbits, but alpha s1-casein and beta-casein were not detectably present in rabbit blood at the examined physiological states.

Amino Acids↗
PubMed1990

Osmotic shock of cultured primary mammary cells amplifies the hormonal induction of casein gene expression.

Primary cells from rabbit mammary gland cultured on floating collagen were transfected with various plasmids in different conditions. Conventional transfection methods using DEAE-dextran or calcium phosphate followed by an osmotic shock with dimethyl sulphoxide (DMSO), polyethylene glycol (PEG) or glycerol did not prevent lactogenic hormones to induce casein synthesis. On the contrary and unexpectedly, casein synthesis was markedly stimulated by transfection. This amplification was obtained as well with DMSO, PEG and glycerol alone or in the presence of DEAE-dextran, calcium phosphate or DNA. None of these compounds induced casein synthesis in the absence of prolactin. A shock by DMSO also amplified the accumulation of beta-casein mRNA in the presence of prolactin. These results show for the first time that primary cultured mammary cells can be efficiently transfected and still keep their capacity to respond to lactogenic hormones. They also indicate that the short osmotic shocks conventionally used in transfection have a potent long-term stimulatory effect on casein gene expression, which is mediated through an unknown mechanism.

Animals↗
PubMed1990

Effects of amiloride on the induction of DNA synthesis and casein gene expression in rabbit mammary explants.

Amiloride, an inhibitor of Na+/H+ exchange, was added at various concentrations to the culture medium of rabbit mammary explants. In the concentration range 100-250 microM, amiloride progessively inhibited 14C-thymidine incorporation induced by insulin, EGF or prolactin. Up to 250 microM, amiloride, which did not inhibit basal protein synthesis, was not cytotoxic, but it reduced basal DNA synthesis at the highest concentration. Addition of amiloride to the culture medium of mammary explants also strongly inhibited the induction of casein synthesis and casein mRNA accumulation by prolactin. The inhibition by amiloride is therefore not specific of the mitogenic action of prolactin since this drug also prevented its lactogenic action. The data reported here describe a new inhibitory action of amiloride on the transmission of the lactogenic signals.

Amiloride↗
PubMed1990

The hormonal control of ovine beta-lactoglobulin gene in cultured ewe mammary explants.

Mammary explants from pregnant ewes were cultured in the presence of insulin, cortisol and prolactin, either alone, or in combination. After 2 d of culture, total RNA was extracted from explants and the content of beta-lactoglobulin mRNA was estimated using a specific labelled cDNA probe. The mRNA for beta-lactoglobulin was only deinduced slowly in the absence of hormone during the culture. Prolactin alone induced the accumulation of the mRNA. Insulin and cortisol added together were also stimulatory, but they only moderately amplified the prolactin effect. Beta-lactoglobulin gene in ewes, is therefore, controlled by the lactogenic hormones which also induce casein gene expression. The amplitude of the stimulation was unexpectedly low. This seems due in part to the fact that the gene was only deinduced weakly. In this respect, beta-lactoglobulin gene appears to be less dependent on lactogenic hormones under these experimental conditions than casein genes.

Animals↗
PubMed1990

The possible involvement of tubulin in transduction of the prolactin signal.

Prolactin has many different biological functions. It stimulates Nb2 cell multiplication through the kinase C transduction mechanism, casein secretion through the phospholipase A2-arachidonic acid-leukotrienes cascade and milk protein gene expression through an unknown mechanism. Colchicine and other tubulin binding drugs inhibit casein gene expression and DNA synthesis stimulated by prolactin whereas chemical compound which alter microtubule without binding tubulin exert no inhibitory effect. Myo-inositol which suppresses some of the colchicine actions in several biological systems does not restore prolactin action after an inhibition by the drug. These data suggest that a tubulin molecule in the vicinity of the prolactin receptor, rather than actual microtubules, is involved in the transduction of the prolactin message from its receptor to milk protein genes.

Animals↗
PubMed1990

Beta-glucan and pectin derivatives stimulate prolactin secretion from hypophysis in vitro.

Previous work has shown that various plant extracts administered to animals stimulate milk protein synthesis through the secretion of prolactin. It has also been shown that beta-glucan and pectin are the active molecules capable of stimulating prolactin release in vivo after intravenous injections. In this work, it is shown that beta-glucan and several pectin derivatives are able to stimulate prolactin secretion from hypophysis fragments incubated for 2 hr in a synthetic medium.

Animals↗
PubMed1990

[Production of human proteins in the blood of transgenic animals].

The human alpha 1-antitrypsin gene has been microinjected into rabbit embryos. A line of transgenic rabbits has thus been established. Human alpha 1-antitrypsin was found in the blood of transgenic animals at the concentration of 1 mg/ml plasma. The human protein was active and separable from its rabbit counterpart. This experiment demonstrates that it is possible to use blood from transgenic animals as a source of recombinant proteins.

Animals↗
PubMed1989

Inhibition of casein synthesis by progestagens in vitro: modulation in relation to concentration of hormones that synergize with prolactin.

We studied the effect of progesterone and its agonist, R 5020, on casein and transferrin production in pregnant rabbit mammary gland explant culture and its modulation by hormones that synergize with prolactin. The glands were obtained from rabbits on days 12-14 of gestation. The progestins had no effect alone, but significantly inhibited ovine and porcine prolactin stimulation of casein synthesis in a dose dependent manner. There were no effects on transferrin content of the tissue, demonstrating a specific effect of progesterone on casein synthesis. In approx 15% of the cultures, prolactin stimulated casein production to very high levels and the progestins lost their inhibitory action. Progestins were also ineffective when the tissue was cultured with prolactin and unphysiologically high levels of insulin (5 mg/l) or cortisol (280 nmol/l), which stimulated casein synthesis to higher levels than prolactin alone. The concentration of cortisol used was 10 times higher than the serum levels seen in rabbits at the stage of gestation studied (approx 10 ng/ml) and corresponded to levels seen at the end of gestation, a period when the glands are secreting milk and progesterone serum levels have commenced to decrease. Thus, when the prolactin effect upon casein synthesis had been potentiated, whether spontaneously or through synergism with insulin or corticoids, progestins were unable to inhibit it, as is the case in lactating tissues. The results show that utilization of unphysiological levels of hormones in culture may distort the response of the tissue, masking responses that are clearly seen in vivo.

Animals↗
PubMed1989

Integration and germ line transmission of foreign genes microinjected into fertilized trout eggs.

Persistence, integration into host genome, germ line transmission and expression of foreign genes microinjected into cytoplasm of fertilized rainbow trout eggs has been examined. Foreign DNA persisted as large random concatenates in approximately 50% of 6 to 12 month-old trout and exhibited a mosaic pattern between tissues. In some cases, free concatenates were observed indicating that extrachromosomal replication occurred in trout. Approximately 50% of the males had the foreign sequences in sperm DNA and all the examined animals transmitted these sequences to their progeny. The percentage of transgenic offsprings ranged from 10 to 30% and putative junction fragments were identified in Southern blot analysis in some of them. These results strongly support the hypothesis that the injected genes became integrated into the genome host, most likely after the first round of chromosomal replication. We also examined the expression of the microinjected plasmids which contained viral or mammalian promoters linked to human or rat growth hormone gene. In no case could exogenous growth hormone be detected.

Animals↗