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Production of human α-lactalbumin (hα-LA) transgenic cloned dairy goats has great potential in improving the nutritional value and perhaps increasing the yield of dairy goat milk. Here, a mammary-specific expression vector 5A, harboring goat β-lactoglobulin (βLG) promoter, the hα-LA gene, neo(r) and EGFP dual markers, was constructed. Then, it was effectively transfected into goat mammary epithelial cells (GMECs) and the expression of hα-LA was investigated. Both the hα-LA transcript and protein were detected in the transfected GMECs after the induction of hormonal signals. In addition, the 5A vector was introduced into dairy goat fetal fibroblasts (transfection efficiency ≈60-70%) to prepare competent transgenic donor cells. A total of 121 transgenic fibroblast clones were isolated by 96-well cell culture plates and screened with nested-PCR amplification and EGFP fluorescence. After being frozen for 8 months, the transgenic cells still showed high viabilities, verifying their ability as donor cells. Dairy goat cloned embryos were produced from these hα-LA transgenic donor cells by somatic cell nuclear transfer (SCNT), and the rates of fusion, cleavage, and the development to blastocyst stages were 81.8, 84.4, and 20.0%, respectively. A total of 726 reconstructed embryos derived from the transgenic cells were transferred to 74 recipients and pregnancy was confirmed at 90 days in 12 goats. Of six female kids born, two carried hα-LA and the hα-LA protein was detected in their milk. This study provides an effective system to prepare SCNT donor cells and transgenic animals for human recombinant proteins.

In this study, we used the Illumina OvineSNP50 BeadChip to conduct a genome-wide association (GWA) analysis for milk production traits in dairy sheep by analyzing a commercial population of Spanish Churra sheep. The studied population consisted of a total of 1,681 Churra ewes belonging to 16 half-sib families with available records for milk yield (MY), milk protein and fat yields (PY and FY) and milk protein and fat contents (PP and FP). The most significant association identified reached experiment-wise significance for PP and FP and was located on chromosome 3 (OAR3). These results confirm the population-level segregation of a previously reported QTL affecting PP and suggest that this QTL has a significant pleiotropic effect on FP. Further associations were detected at the chromosome-wise significance level on 14 other chromosomal regions. The marker on OAR3 showing the highest significant association was located at the third intron of the alpha-lactalbumin (LALBA) gene, which is a functional and positional candidate underlying this association. Sequencing this gene in the 16 Churra rams of the studied resource population identified additional polymorphisms. One out of the 31 polymorphisms identified was located within the coding gene sequence (LALBA_g.242T>C) and was predicted to cause an amino acid change in the protein (Val27Ala). Different approaches, including GWA analysis, a combined linkage and linkage disequilibrium study and a concordance test with the QTL segregating status of the sires, were utilized to assess the role of this mutation as a putative QTN for the genetic effects detected on OAR3. Our results strongly support the polymorphism LALBA_g.242T>C as the most likely causal mutation of the studied OAR3 QTL affecting PP and FP, although we cannot rule out the possibility that this SNP is in perfect linkage disequilibrium with the true causal polymorphism.

The ontogeny of the immune system and the effect thereon by type of infant feeding is incompletely understood. We analyzed frequencies and composition of immune cells in blood of breastfed (BF) and formula-fed (FF) infants at 1.5, 4, and 6 mo of age. Three formulas with the same protein concentration but with varying levels of alpha-lactalbumin and caseinoglycomacropeptide were compared. Twenty-nine exclusively BF infants served as reference, and 17 infants in each formula group completed the study. Whole blood and PBMCs were analyzed by flow cytometry and immunoflow cytometry, respectively. Leukocyte count of BF infants increased with time due to increased frequency of neutrophils. Lymphocyte count was high at 1.5 mo and was unchanged over time, as were the relative proportions of CD4+ alphabetaT cells, CD8+ alphabetaT cells, B cells, NK cells, and gammadeltaT cells. Most CD45R0+CD3+ cells were HLA-DR- and hence memory cells. Compared with breastfeeding, formula feeding resulted in a significant decrease in proportion of NK cells, but a significant increase in naive CD4+ alphabetaT cells and an elevated CD4-to-CD8 ratio, that is, 3.3 in the combined FF groups compared with 2.6 in the BF group. No significant differences were found between the three groups of FF infants. In conclusion, blood cells of lymphoid lineage did not change significantly in frequencies or composition from 1.5 to 6 mo of age in BF infants. In contrast, FF infants displayed an ongoing maturation of adaptive immunity cells and a delayed recruitment of innate immunity cells as compared with BF infants.

The analysis of transgene inheritance is an important step in the molecular and genetic characterization of transgenes. In this manuscript, two approaches to characterize the inheritance of transgenes are described. The first approach is based on the expression of the transgene phenotype and the second is based on the analysis of transgene DNA. Instructions on how to make crosses and develop breeding populations are outlined and the importance of these breeding populations in the analysis of transgene inheritance is explained. The number of individuals needed to determine segregation ratios and the statistic used to test these ratios are described. Examples of inheritance patterns that deviate from known expectations are provided and the possible causes of these deviations are discussed.

We studied the transcription of selected genes involved in the regulation of cell turnover during milk stasis and lactation rescue in individual mammary glands of sows and evaluated the timeframe for lactation rescue and the subsequent productivity of rescued glands. Suckling was prevented in two glands from each of five sows by taping them the day after farrowing for 1 or 3 d (Closed 24 h and Closed 72 h glands, respectively). After removing the tape, Closed 24 h glands were suckled until weaning. Closed 72 h glands were refused by the piglets and therefore remained unsuckled. Mammary gland biopsies were collected to quantify the transcription of genes involved in cell turnover and the percentage of proliferating and apoptotic cells. Transcriptional data were analyzed with a gamma-distributed generalized linear mixed model. Mammary transcription of alpha-lactalbumin was high in suckled glands the day after farrowing, indicating onset of lactation, but was downregulated after 1 d of milk stasis. The prolactin receptor mRNA was downregulated and IGFBP-5 mRNA was upregulated within 1 d of milk stasis. The downregulation of alpha-lactalbumin and prolactin receptor and upregulation of IGFBP-5 mRNA noted after 1 d of milk stasis was maintained in Closed 72 h glands (those glands regressed) but reversed on d 4 and 6 of lactation in Closed 24 h glands. Mammary IGF-I mRNA was not regulated in response to milk stasis or lactation rescue. The percentage of proliferating cells in mammary glands was high prepartum (13.1%) and intermediate (7.8%) the day after farrowing. By d 6 of lactation, the percentage of proliferating cells was increased to 10.1% (P < 0.01) in glands suckled regularly but decreased to 5.9% (P < 0.05) in regressing glands (Closed 72 h glands). Glands rescued after 1 d of milk stasis had lower productivity throughout lactation than glands suckled regularly, as indicated by the BW of piglets suckling these glands (242 vs. 315 g/d, respectively; P < 0.05). In conclusion, regularly suckled glands had a greater cell proliferation, greater transcriptions of alpha-lactalbumin and prolactin receptor genes, and less IGFBP-5 transcription compared with rescued (Closed 24 h) and regressing (Closed 72 h) glands. Glands that were not suckled for 1 d could be rescued, although their subsequent productivity was lower, whereas glands not suckled for 3 d could not be rescued.

Yeast artificial chromosomes (YACs) as transgenes in transgenic animals are likely to ensure optimal expression levels. Microinjection of YACs is the exclusive technique used to produce YACs transgenic livestock so far. However, low efficiency and high cost are its critical restrictive factors. In this study, we presented a novel procedure to produce YACs transgenic livestock as mammary gland bioreactor. A targeting vector, containing the gene of interest-a human serum albumin minigene (intron 1, 2), yeast selectable marker (G418R), and mammalian cell resistance marker (neo(r)), replaced the alpha-lactalbumin gene in a 210kb human alpha-lactalbumin YAC by homogeneous recombination in yeasts. The chimeric YAC was introduced into goat fetal fibroblasts using polyethylene glycol-mediated spheroplast fusion. PCR and Southern analysis showed that intact YAC was integrated in the genome of resistant cells. Perhaps, it may offer a cell-based route by nuclear transfer to produce YACs transgenic livestock.

In this study, we investigated the cellular and molecular events involved in parity-related alterations in mammary gland (MG) proliferation and differentiation. Rat MGs were removed on day 9 of either first (nulliparous), second (primiparous) or third (multiparous) pregnancy. Expression of steroid hormone receptors along with cellular biomarkers of proliferation and differentiation were quantified in all MG tissue compartments by immunohistochemistry. Wnt-4 (a Wingless-like morphogenic gene involved in MG development), ERbeta and ERbeta2 mRNA were evaluated by RT-PCR analysis. Serum levels of mammotrophic hormones were measured. In comparison to nulliparous and primiparous rats, multiparous animals exhibited decreased luminal cell proliferation and PR levels, whereas alpha-lactalbumin, ERalpha, ERbeta and ERbeta2 expression were increased. In myoepithelial cells, while parity induced a decrease in proliferative activity, subsequent pregnancies and lactations lead to an increased state of differentiation. Our results showed that at least two periods of pregnancy and lactation were necessary to modify the studied parameters. The lower proliferative activity and higher differentiation state of the multiparous MG are associated with both a decreased PR expression and increased ERalpha and ERbeta expression. Since ERbeta and/or ERbeta2 isoform expression was related to parity history, results suggest that the decreased proliferative activity and PR expression observed in the MG of multiparous animals may be associated with overexpression of ERbeta and/or the ERbeta2 isoform, thereby antagonizing the proliferative effects associated with ERalpha.

Nitric oxide (NO) enhances prolactin-stimulated DNA synthesis and inhibits prolactin-induced differentiation in mouse mammary epithelium. The molecular pathways used by NO were determined by employing specific inhibitors of the transducers utilized by NO. Inhibitors of the Jun N-terminal kinase (JNK) blocked the effect of NO on DNA synthesis, although this appeared to involve a protein kinase G (PKG)-independent pathway. In contrast, inhibitors of the extracellular signal-regulated kinase (ERK) prevented NO from suppressing alpha-lactalbumin accumulation and this effect was PKG-dependent. NO can also elevate cAMP through the inhibition of phosphodiesterase 3 and cAMP mimicks the actions of NO on both DNA synthesis and differentiation. However, suppression of cAMP levels did not prevent the effects of NO. Therefore, NO uses two separate pathways to affect mammary epithelium: it stimulates growth via JNK and inhibits differentiation through ERK.

Lactose synthase (a complex of beta1,4-galactosyltransferase and alpha-lactalbumin) forms lactose in the Golgi complex of mammary epithelial cells. To determine whether alpha-lactalbumin is a limiting component in this complex, transgenic mice that expressed bovine alpha-lactalbumin were studied. Transgenic mice produced 0.5 to 1.5 mg/ml of bovine alpha-lactalbumin in their milk, 5- to 15-fold more alpha-lactalbumin than in milk of control mice. Transgenic and control mice produced milk with the same concentrations of lactose, cream, and total solids, and showed similar mammary gland growth, morphology, and histology. Milk from transgenic mice had 0.6% less protein than milk from control mice (P < 0.05). The in vitro lactose synthase activity in mammary gland homogenates from alpha-lactalbumin transgenic mice was increased (P < 0.05), demonstrating that bovine alpha-lactalbumin could interact with murine beta1,4-galactosyltransferase. Pups reared by lactating transgenic mice showed a 4% increase in growth on d 10 of lactation, suggesting that milk production was increased (P = 0.06). Milk volume, estimated using the weigh-suckle-weigh technique, tended to be higher (although not significantly) in transgenic mice (P = 0.11). These results suggest that augmenting alpha-lactalbumin expression in the dam increases the growth of suckling offspring.

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.

Transgenic technology permits major modifications of phenotype by introducing subtle changes in genotype. For domestic farm species, genetic modification may be used to enhance agricultural production or to generate novel genotypes capable of producing heterologous proteins for biomedical applications. The advent of in vitro embryo production techniques has facilitated the large-scale, commercial use of transgenic technology in cattle. Accordingly, we employed in vitro-produced zygotes and embryos in an effort to generate transgenic cattle. Overall, pronuclei in 36,530 in vitro matured and fertilized zygotes were microinjected with a construct designed to express human alpha-lactalbumin in the mammary gland. Of these, 1,472 developed and were transferred to recipients, including 148 twin transfers. Initial pregnancy rate on Day 30 of gestation was 28% (374/1,324). Subsequent calving rate was 17% (226/1,324). Eighteen calves (8%) were transgenic. In vitro produced embryos were used to facilitate breeding of transgenic bulls. Frequency of transgene transmission varied from 3 to 54% between bulls, indicating varying degrees mosaicism. Embryos produced in vitro by these bulls were biopsied and screened for transgenesis prior to transfer to recipients; so far all (6/6) calves born from screened, transgenic embryos were themselves transgenic.

Plants are particularly suitable bioreactors for the production of proteins, as their eukaryotic nature frequently directs the appropriate post-translational modifications of recombinant proteins to retain native biological activity. The autotrophic growth of plants makes this in vivo biosynthesis system economically competitive for supplementation or replacement of conventional production systems in the future. For the production of biologically active proteins, food plants provide the advantage of direct delivery via consumption of transformed plant tissues. Here we describe the production of recombinant human milk proteins in food plants for improvements in human nutrition and health, with emphasis on enhanced nutrition for non-breast fed infants as well as children and adults. Nutritional improvements in edible plants generated through advancements in recombinant DNA technology are rapidly repositioning the world for enjoyment of a more healthful diet for humans in all age groups.

High production of milk and its components are necessary to allow maximal growth of developing pigs. In this study, transgenic pigs were produced containing the alpha-lactalbumin gene, whose product is a potential limiting component in the production of milk. Two lines of transgenic pigs were produced to analyze the effects that overproduction of the milk protein alpha-lactalbumin may have on milk production and piglet growth. Transgenic pigs were produced through microinjection of the bovine alpha-lactalbumin gene. The gene construct contained 2.0 kb of 5' flanking region, the 2.0 kb coding region, and 329 bp of 3' flanking region. Sows hemizygous for the transgene produced as much as .9 g of bovine alpha-lactalbumin per liter of pig milk. The production of the bovine protein caused approximately a 50% increase in the total alpha-lactalbumin concentration of pig milk throughout a lactation. The concentration of bovine alpha-lactalbumin was highest on d 0 and 5 of lactation and decreased as lactation progressed. The ratio of bovine to porcine alpha-lactalbumin changed during the sow's lactation. This ratio was 4.3 to 1 on d 0 of lactation, but by d 20 of lactation the ratio was .43 to 1. This suggested that the bovine transgene and the endogenous porcine gene are under slightly different control mechanisms. The higher level of total alpha-lactalbumin present on d 0 of lactation was correlated with higher lactose percentage on d 0 in transgenic sows (3.8%), compared with controls (2.6%) (P < .01). Although there was also a trend for higher lactose percentage in transgenic sows on d 5 and 10 of lactation, no significant differences were observed. These data suggest that alpha-lactalbumin is limiting early in lactation of swine. Furthermore, higher concentrations of alpha-lactalbumin early in lactation may boost milk output.

A recombinant bovine alpha-lactalbumin, possessing an additional N-terminal methionyl residue, was expressed in Escherichia coli. In order to address the effects of the N-terminal methionyl residue on conformational stability, the thermal stability of the recombinant alpha-lactalbumin was investigated by measuring temperature-dependence of circular dichroism spectra, and it was compared with that of authentic alpha-lactalbumin from bovine milk. The thermal stability of the recombinant alpha-lactalbumin was significantly lower than that of authentic alpha-lactalbumin. The enthalpy change of unfolding of the recombinant protein was found to be the same as that of the authentic one when compared at the same temperature. Therefore, the N-terminal methionyl residue seems to destabilize the conformation of recombinant alpha-lactalbumin through some entropic effects.

Bovine alpha-lactalbumin (alpha-LA) and bovine beta-casein (beta-CN), from milk from transgenic mice were characterized and partially purified using electrophoretic, immunoblotting, and chromatographic methods. The transgenically expressed bovine milk proteins were identified using PAGE or by a combination of preparative isoelectrofocusing followed by Western immunoblotting. The heterologous bovine alpha-IA and bovine beta-CN had molecular masses that were identical to those of those of the native proteins. The estimated expression of the proteins was 1.0 mg/ml of milk for alpha-LA and 3.0 mg/ml for beta-CN. The calcium binding of bovine alpha-LA suggested that the protein produced in murine milk has the same electrophoretic shift as native bovine alpha-LA after the removal of calcium. Nitrogen-linked glycosylation of native and murine synthesized bovine alpha-LA was identified by peptide-N-glycosidase F treatment, and the N-terminal amino acid sequence of HPLC-purified bovine alpha-LA from mouse milk was confirmed to be identical to native bovine alpha-LA. In addition, the phosphorylation of the bovine beta-CN expressed in the milk of transgenic mice was the same as that of native bovine beta-CN, as determined by phosphatase digestion.

The level of expression of transgenes in transgenic animals varies among lines, and is often much lower than that of endogenous genes (position effects). In order to surmount position effects and establish a more efficient production system of transgenic animals producing pharmaceutical proteins in their milk, transgenic rats carrying 210-kb YAC DNA containing the human alpha-lactalbumin gene were produced. Three transgenic lines transmitted the transgene to the next generation. They had one copy of the alpha-lactalbumin gene and secreted human alpha-lactalbumin in their milk at concentrations of 2.0-4.3 mg/ml. No position effect was seen. The transgene was expressed specifically in the mammary gland of the transgenic rats. The 210-kb region is thought to contain all the DNA elements required for proper expression of the human alpha-lactalbumin gene. The YAC carrying the human alpha-lactalbumin gene is a potential vector for the expression of foreign genes in the mammary gland.

Cellular DNA, milk protein content, and protein secretion by bovine mammary explants were compared to cultures of confluent and growing primary bovine mammary secretory cells over 4 d. Explants were obtained at slaughter from eight Holstein cows (120 +/- 35 d lactation). Primary cells were grown to confluence, cryopreserved, thawed, and cultured through five passages. Explants and cells were cocultured with liver and adipose tissue in the presence of somatotropin, insulin-like growth factor-I, and somatotropin + insulin-like growth factor-I. Cellular DNA and milk proteins were assayed using fluorescent probes and flow cytometry. Media proteins were assayed by densitometer scanning of electrophoresis gel bands. DNA content of explant, confluent, and growing primary cells increased similarly through the 96 h incubation. DNA content in G0G1 phase was increased by: (a) insulin-like growth factor-I in explant cells; (b) somatotropin, insulin-like growth factor-I, and their combination in confluent primary cells; and (c) the combination of somatotropin and insulin-like growth factor in growing primary cells. Approximately 65% of explant and confluent primary cells were in the G0G1 or differentiated phase compared to 47% for the growing primary cells. Whey protein content and secretion were similar among cell types. Explant cells contained and secreted more beta-casein than primary cells but secretion trends for beta-casein and k-casein were similar after 48 h for both cell types. Results suggest that primary cell cultures are comparable to explant cultures when used to study mechanisms of DNA and milk protein synthesis and secretion.

Explants of mammary tissue from cows in late pregnancy were incubated for 72 h in serum-free, hormonally defined media to investigate the regulation of the bovine mammary IgG1 receptor. Treatments included incubation in basal medium alone, basal medium plus estradiol-17 beta, basal medium plus prolactin, or basal medium plus estradiol-17 beta and prolactin. alpha-Lactalbumin production was measured by radioimmunoassay in culture supernatants collected at 24, 48, and 72 h. Explants were examined immunohistochemically for expression of the IgG1 receptor at 24, 48, and 72 h. alpha-Lactalbumin concentrations increased, and IgG1 receptor expression decreased, by 72 h with explants cultured in the medium containing prolactin. Results suggest that, in addition to its positive lactogenic effect, prolactin decreases expression of the bovine mammary IgG1 receptor.

The milk of livestock can be modified dramatically by introducing foreign DNA into the germline. Exclusive expression of this DNA is ensured by the presence of regulatory sequences from mammary gland-specific genes. In sheep > 50% of the protein in milk can be encoded by a transgene and it appears that the foreign protein is additional to the normal complement of proteins. However, many technical hurdles (DNA configuration, low efficiency of transgenesis, and transgene stability) still prevent the routine use of this technology. In addition, such milk products have not yet received regulatory approval. These difficulties are not insurmountable. Transgenic methods can also be used to study the molecular basis of milk biogenesis. The effect on milk production in mice with all endogenous alpha-lactalbumin genes removed and in mice in which the murine genes were then replaced with human homologues is described. alpha-Lactalbumin, and consequently lactose, is essential for normal milk formation, and the human gene is expressed more efficiently than the murine gene.

To evaluate whether estrogen hastened involution of mammary tissue, Holstein cows were injected with 4 ml of ethanol excipient (n = 21) or 15 mg of estradiol-17 beta (n = 23) on each of the 4 d that preceded final milk removal. Dates of final milk removal (d 0) were designated as 60 d prior to expected dates of calving. Milk volumes were recorded, and samples were collected prior to the first and fourth injections. During the dry period, each mammary quarter within the cow was sampled once to collect secretions on dates that corresponded to d 0, 3, 11, and 25 or 1, 7, 18, and 30 of the dry period. Milk synthesis and secretion declined abruptly because of treatment. The decreased concentrations of alpha-lactalbumin, lactose, citrate, and potassium in secretions of controls, as well as the increased somatic cells, protein, lactoferrin, and sodium, occurred earlier in secretions from treated cows. These shifts of approximately 6 d, relative to days dry, suggested that exogenous estradiol increased the involution rate of mammary tissue.

Studies were carried out to determine the possible mechanism(s) by which prolactin (PRL) stimulates alpha-lactalbumin and galactosyl transferase activity in cultured mouse mammary tissues. In earlier studies it was shown that the onset of the PRL stimulation of galactosyl transferase activity occurs between 4 and 8 hr after adding prolactin to cultured mouse mammary gland explants, and a maximum effect is evoked by 24 hr. In contrast, an effect of prolactin on alpha-lactalbumin activity occurred 24 hr after adding PRL to mammary gland cultures, but not at earlier times. In the present studies, it is shown that prolactin effects an increased tissue accumulation of mRNAs for both alpha-lactalbumin and galactosyl transferase after a 4 to 6 hr culture with prolactin. The lowest concentration of prolactin that stimulates the accumulation of both galactosyl transferase and alpha-lactalbumin mRNA is approximately 10 ng/ml, and a maximum response is achieved with 200 ng/ml. Thus, it is likely that the effect of PRL on the activity of galactosyl transferase is causally associated with the effect of prolactin on the accumulation of its mRNAs. However, the PRL stimulation of alpha-lactalbumin activity is not temporally associated with the tissue accumulation of the mRNA for alpha-lactalbumin.

Mice carrying either a deletion of the murine alpha-lactalbumin (alpha-lac) gene (null allele) or its replacement by the human alpha-lac gene (humanized allele) have been generated by gene targeting. Homozygous null females are alpha-lac-deficient, produce reduced amounts of thickened milk containing little or no lactose, and cannot sustain their offspring. This provides definitive evidence that alpha-lac is required for lactose synthesis and that lactose is important for milk production. Females homozygous for the humanized allele lactate normally, indicating that human alpha-lac can replace murine alpha-lac. Mouse and human alpha-lac expression was compared in mice heterozygous for the humanized allele. The human gene expressed approximately 15-fold greater mRNA and approximately 14-fold greater protein than the mouse, indicating that the major determinants of human alpha-lac expression are close to, or within, the human gene and that the mouse locus does not exert a negative influence on alpha-lac expression. Variations in alpha-lac expression levels in nondeficient mice did not affect milk lactose concentration, but the volume of milk increased slightly in mice homozygous for the humanized allele. These variations demonstrated that alpha-lac expression in mice is gene dosage dependent.

Polyclonal antibodies to mouse alpha- and beta/gamma-caseins were raised in rabbits. These antibodies display tissue- and species specificity as shown by immunoblotting. Immunohistochemical analyses demonstrate that both alpha- and beta/gamma-caseins were synthesized and secreted from virtually all lactating mammary epithelial cells, in a pattern very similar to that of the mouse alpha-lactalbumin. Residual amounts of caseins were located also in the apical surface of epithelial cells surrounding the ducal lumen of virgin mammary gland sections. In contrast to the significant level of gamma-casein in the milk, the amount of this protein compared to alpha- or beta-caseins was extremely low in medium conditioned for 24 h by mammary explants of mid-pregnant mice immediately after explantation or after 4 days.

alpha-Lactalbumin is an abundant milk-specific calcium metalloprotein which has an evolutionary relationship to lysozyme. It modifies the substrate specificity of a Golgi galactosyltransferase by forming the lactose synthetase binary complex. Lactose, together with other sugars and diffusible ions, is responsible for the osmotic pressure of milk. To assess the involvement of alpha-lactalbumin in lactogenesis, alpha-lactalbumin-deficient mice were created by disrupting the gene by homologous recombination in embryonic stem cells. Homozygous mutant mice are viable and fertile but females cannot feed their offspring. They produce a highly viscous milk that pups appear to be unable to remove from the mammary gland. This milk is rich in fat and protein and is devoid of alpha-lactalbumin and lactose. The phenotype of heterozygous mice was found to be intermediate, with a 40% decrease in alpha-lactalbumin but only a 10-20% decrease in the lactose content of their milk compared with wild-type animals. These results emphasize the key function of alpha-lactalbumin in lactogenesis and open new opportunities to manipulate milk composition.

Mammary tissue from pigs on days 60, 80, 90, 100 and 100+ (days 106-111) of pregnancy has been cultured in vitro as explants. The total accumulation in tissue and culture medium of the whey proteins alpha-lactalbumin and beta-lactoglobulin has been measured using specific radioimmunoassays. The control, uncultured tissue showed progressive morphological development from sparse, non-secretory epithelial tissue on day 60 to full lobulo-alveolar development with some accumulated secretion from day 100. In uncultured explants beta-lactoglobulin could be detected consistently from day 90 (13 +/- 12 ng/micrograms DNA, n = 4) and alpha-lactalbumin from day 100 (1.3 +/- 0.5 ng/micrograms DNA, n = 11). At all stages of pregnancy, both whey proteins increased markedly during the period of culture (up to 7 d). Stimulation of alpha-lactalbumin appeared to be primarily under prolactin control. Prolactin increased alpha-lactalbumin accumulation to a similar extent alone, or in the presence of insulin and/or corticosterone. The response to prolactin was dose-dependent over the range 0.4-20 nM (10-500 ng/ml). Porcine prolactin was more potent than ovine prolactin. There was no effect of porcine growth hormone and no synergism detected between prolactin and tri-iodothyronine. By contrast, no specific hormonal requirements were established for accumulation of beta-lactoglobulin, which appeared to increase in vitro if tissue remained viable in various combinations of insulin, corticosterone and prolactin. It was not stimulated by growth hormone. There was some indication of a prolactin-sensitive component in longer term cultures after day 4.

The mouse alpha-lactalbumin gene has been replaced with the human gene by two consecutive rounds of gene targeting in hypoxanthine phosphoribosyltransferase (HPRT)-deficient feeder-independent murine embryonic stem (ES) cells. One mouse alpha-lactalbumin allele was first replaced by an HPRT minigene which was in turn replaced by human alpha-lactalbumin. The end result is a clean exchange of defined DNA fragments with no other DNA remaining at the target locus. Targeted ES cells at each stage remained capable of contributing efficiently to the germ line of chimeric animals. Double replacement using HPRT-deficient ES cells and the HPRT selection system is therefore a powerful and flexible method of targeting specific alterations to animal genes. A typical strategy for future use would be to generate a null mutation which could then be used to produce multiple second-step alterations at the same locus.

A four-fold increment of alpha-lactalbumin mRNA was induced in mammary gland explants from mid-pregnant Balb/c mice cultured for 24h in media containing insulin, cortisol and T3, and this response was enhanced by 40% with the addition of prolactin. In contrast, elevated beta-casein gene expression was evident only in explants cultured in media with insulin, cortisol, T3 and prolactin. These results suggest that the induction of the alpha-lactalbumin gene was prolactin-independent and did not result from retention of endogenous prolactin, or its effects, by the mammary explants.

The propeptides of lysosomal enzymes have been implicated in membrane association and mannose 6-phosphate-independent sorting to the lysosome (Rijnboutt, S., Aerts, H., Geuze, H. J., Tager, J. M., and Strous, G. J. (1991) J. Biol. Chem. 266, 4862-4868; McIntyre, G. F., and Erickson, A. H. (1991) J. Biol. Chem. 266, 15438-15445). In this report, the function of the propeptide of procathepsin D in sorting to the lysosome was directly assessed using a cathepsin D deletion mutant lacking the propeptide, and using a chimeric cDNA encoding the cathepsin D propeptide fused to the secretory protein alpha-lactalbumin. Proteins encoded by these cDNAs were expressed in mouse Ltk- cells and in human hepatoma Hep G2 cells, and then immunoprecipitated and analyzed by SDS-polyacrylamide gel electrophoresis. The deletion mutant was glycosylated but was rapidly degraded in a chloroquine-independent fashion and did not assume an active conformation. Thus the propeptide appeared to be necessary for correct folding. The chimeric protein was glycosylated and secreted. The coincidence of complex oligosaccharide modification and secretion of the chimeric protein suggested that it was slowly released from the endoplasmic reticulum and rapidly passed through the cell to the extracellular compartment. This was confirmed by immunofluorescent localization of the proteins. The data indicated that the propeptide appeared to be necessary for folding of cathepsin D but, unlike the yeast vacuolar propeptides, was not sufficient to direct a secretory protein to the lysosome in fibroblasts or in epithelial cells.

An expression plasmid for goat alpha-lactalbumin in Saccharomyces cerevisiae, pSKA100, was constructed into a shuttle vector, pYG100, by inserting cDNA which encodes goat pre-alpha-lactalbumin and two-thirds of the 3'-non-coding region. The goat alpha-lactalbumin was expressed under the yeast glyceraldehyde 3-phosphate dehydrogenase (GPD) promoter and terminator of pYG100 and secreted in the growth medium for yeast as a precise mature protein, possessing specific activity essentially the same as that of authentic goat alpha-lactalbumin in lactose synthesis.

A cDNA encoding the mature region of goat alpha-lactalbumin and the 3'-non-coding region was fused to cDNA of the N-terminal half of porcine adenylate kinase which had been placed under the control of the tac promoter in an expression vector in Escherichia coli. In addition, a methionine codon was inserted between the two cDNAs. When the plasmid carried the full-length 3'-non-coding region, little accumulation of the fused protein was observed. However, the deletion of two-thirds of the 3'-non-coding region produced significant expression of the fused protein in E. coli strain JM105. Since goat alpha-lactalbumin contains no methionine residue, the mature goat alpha-lactalbumin was isolated by CNBr digestion of the fused insoluble protein and refolded using thioredoxin. The homogeneous and biologically active goat alpha-lactalbumin was purified by Ca2+ ion-dependent hydrophobic chromatography.

The gene for alpha-lactalbumin has been cloned from a tammar wallaby (Macropus eugenii) mammary gland cDNA library. Tammar alpha-lactalbumin has approximately 50 and 30% homology to the alpha-lactalbumins of eutherians at the levels of nucleotide and protein sequence respectively. Comparison of the inferred tammar polypeptide sequence with the sequence of the eutherian proteins reveals extensive divergence at almost all of the non-essential amino acid residues. However, the hydropathy plots of the tammar protein are almost identical to those of eutherian alpha-lactalbumins, suggesting that protein conformation is conserved. The tammar gene encodes a transcript of approximately 975 bases. Northern blot analysis of hormone-stimulated mammary gland explants shows that maximal induction of alpha-lactalbumin mRNA is dependent on prolactin and that expression is not modulated by other hormones that play a role in the initiation of lactation in eutherians.

Stiulation of milk-protein synthesis in mouse mammary epithelium in vitro requires insulin, hydrocortisone, and prolactin. The requirement for hydrocortisone can be replaced by spermidine. The possibility that spermidine mediates the effect of glucocorticoid is also supported by the observation that the cellular spermidine concentration increases before the accelerated synthesis of casein and alpha-lactalbumin.

In organ cultures of mammary glands from mice in midpregnancy, addition of both insulin and prolactin induces a marked accumulation of alpha-lactalbumin, whereas the augmentation of casein synthesis requires the presence of insulin, prolactin, and cortisol. Addition of 0.5 mM dibutyryl cyclic AMP resulted in complete inhibition of alpha-lactalbumin accumulation and partial inhibition of casein synthesis. Furthermore, either cholera toxin at 0.1-1.0 microgram/ml (a stimulator of adenylate cyclase) or 3-isobutyl-1-methylxanthine (an inhibitor of phosphodiesterase) in combination with 2 mM cyclic AMP, produced a similar pattern of inhibition of alpha-lactalbumin and casein synthesis in cultured tissue. During culture of mammary explants in medium containing no hormone, or insulin alone, or insulin, prolactin, and cortisol, the tissue content of cyclic AMP decreased rapidly, reaching half the initial level in 24-48 hr. These results indicate that cyclic AMP plays "negative" regulatory function in hormonal induction of milk protein synthesis during the development of the mammary gland.

The amino acid requirement of rat mammary cells for milk protein synthesis was investigated in dispersed cell culture. A three-dimensional central composite design utilizing three variables (X1 = lysine; X2= methionine, valine, and arginine; X3 = isoleucine, tryptophan, threonine, phenylalanine, and histidine) at five concentrations each, was duplicated twice with mammary cells from lactating Sprague-Dawley rats. The optimum combination of amino acids for maximum milk protein synthesis from multiple regression models was X1 15.0-, X2 4.5-, and X3 1.5-fold their quantities in Eagle's minimal essential medium with leucine, tyrosine, cystine, and glutamine at the base 1-fold in the medium.

Rat alpha-Lactalbumin (alpha-LA) mRNA has been purified from the total RNA of 4-day lactating mammary gland by poly(U)-Sepharose chromatography and by fractionation of the poly(A)+ RNA on sucrose gradients. alpha-LA mRNA was homogeneous as judged by electrophoresis in urea/agarose gel and in 1.5% agarose gel under glyoxal-denaturation conditions, which gave a molecular weight of 210,000. The chymotryptic fingerprints of the protein synthesized with this mRNA, in a translational system derived from wheat germ, were similar to those of purified rat alpha-LA. In rat milk or lactating gland, the content of Mr 42,000 casein is 6 times greater than that of Mr 29,000 casein and 10 times greater than that of alpha-LA. However, alpha-LA mRNA (Mr 210,000) and two casein mRNas (Mr 460,000 and 390,000), coding for Mr 42,000 and 29,000 caseins, were present in equal proportions when measured with cDNA probes in the total RNA of 4- to 5-day lactating gland. Moreover, alpha-LA and total casein were synthesized in a ratio fo 1:2.5 in a wheat-germ translational system using the total RNA of the lactating gland. The results suggest post-transcription controls in the synthesis of these proteins.

Primary mammary carcinomas induced in nonlactating rats by 7,12-dimethylbenz(alpha)anthracene or methylnitrosourea contained alpha-lactalbumin (alphaLA) in quantities equal to or less than 10% of the amounts found in the parenchyma of the 5-day lactating gland. Only two of five transplantable mammary carcinomas contained alphaLA when growing in rats without hormonal stimulation. Hormonal stimulation maintained by transplantation into lactating females for 146 generations (65 months) failed to induce alphaLA production in dimethylbenz(alpha)anthracene no.1 transplantable mammary carcinoma. Transplantation of a pituitary gland under the kidney capsule of the host (a) increased alphaLA content of primary dimethylbenz(alpha)anthracene-induced mammary carcinomas, (b) reduced alphaLA content of primary methylnitrosourea-induced mammary carcinomas, and (c) was unable to modify the alphaLA levels in five transplantable mammary carcinomas.

1. The expression of alpha-lactalbumin and casein genes was examined in guinea-pig mammary tissue taken from animals both pre- and post-partum. 2. Analysis of total RNA by RNA excess hybridization with sequence-specific complementary DNA probes demonstrated that alpha-lactalbumin mRNA was present late in pregnancy, and that maximum concentrations were present at parturition. Casein gene transcripts were absent late in pregnancy (62 days), but by parturition were present at concentrations identical to those found at all time points examined throughout lactation. 3. Studies using mammary explants in organ culture showed that tissue from pregnant animals, or animals at parturition, synthesized and secreted only alpha-lactalbumin. After parturition, at the onset of casein synthesis, differential rates of secretion of alpha-lactalbumin and the caseins were observed. 4. The results are discussed in terms of the multiple intracellular mechanisms involved in the regulation of milk protein gene expression in the guinea-pig mammary gland.

The interplay of insulin, glucocorticoid, and prolactin induces differentiation of mouse mammary gland in culture. Earlier studies indicated that spermidine plays a key role in hormonal control of mammary development by mediating some of the lactogenic actions of glucocorticoid. In this study we examined the effect of progesterone, an inhibitor of lactogenesis, on milk protein synthesis and spermidine accumulation. The maximal degree of inhibition, which was attained at 300 nM, was about 40% for casein synthesis, 90% for alpha-lactalbumin synthesis, and 70% for spermidine accumulation. The increase in the activity of S-adenosyl-L-methionine decarboxylase and spermidine synthetase, two key enzymes in polyamine biosynthesis, was inhibited by 55 and 65%, respectively, by progesterone. The extent of progesterone inhibition of milk protein synthesis was greatest in cultured tissue from virgin mice, which contained a low level of spermidine, whereas mammary explants from lactating mice, which contained a high level of spermidine, were insensitive to progesterone. The inhibitory effect of progesterone can be partially antagonized by cortisol, which causes an enhancement of spermidine accumulation. These results suggest that the synthesis and accumulation of spermidine serve as targets for the inhibitory action of progesterone on lactogenesis.

A comprehensive listing of putative human breast carcinoma cell lines and the extent to which each has been characterized is presented. Criteria used to certify the human, mammary, and malignant origin of a cell line include: (a) a reliable histopathological diagnosis; (b) interspecies specificity established by human karyotype, isoenzyme profiles, and/or cell surface antigenicity; (c) intraspecies specificity, demonstrated by genetic evidence of a unique, human donor distinct from other cells including HeLa cells; and (d) organ specficity, supported by morphological evidence of epithelial structure and secretory activity, and especially by the expression of differentiated functions; these include presence of receptors for sex steroid hormones, hormone responsiveness, and production of milk proteins, fatty acids, or milk-specific antigens. Of the 47 cell lines for which data are here reported, 22 have been shown to be derived from human non-HeLa donors and to have epithelial morphology as revealed by light or electron microscopy. Differentiated function has been recorded for 19 cell lines. Additional human breast cancer cell lines have been reported, but characterization of some of these has been insufficient to judge the legitimacy of their predigrees. For others mammary origin is questionable. Six purported breast cell lines are in reality HeLa cells, and one is of nonhuman origin.

Mammary explants from both pregnant and virgin rats cultured in medium containing insulin, hydrocortisone and prolactin show a progressive increase in alpha-lactalbumin activity. However, when cultured with insulin and hydrocortisone only, explants from pregnant, but not those from virgin rats show an induction of alpha-lactalbumin-like activity similar to that seen when prolactin is present. The activity induced in the absence of exogenous prolactin corresponds to newly synthesized alpha-lactalbumin molecules, since 1) the activity is suppressed completely by rabbit anti-rat alpha-lactalbumin serum and 2) culture in the presence of [3H]-amino acids generates [3H]-alpha-lactalbumin, identified by SDS-polyacrylamide gel electrophoresis. Mammary tissue from virgin rats can be rendered responsive to insulin and hydrocortisone in this regard by administration in vivo of either progesterone or estrogen, but not by desoxycorticosterone acetate. This ability to convert virgin rat mammary cells to a state in which they are independent of exogenous prolactin in terms of alpha-lactalbumin synthesis represents a heretofore unreported effect of the female sex steroids.

Cultures of normal rat mammary cells have been developed which produce alpha-lactalbumin (a-LA) for several months in response to physiological levels of corticosterone whereas none is measureable without glucocorticoid. In contrast to recent studies in short-term organ cultures, these long-term cell cultures derived from virginal or mid-pregnant rats show no inhibition of a-LA production at high glucocorticoid concentrations. Comparable a-LA production was observed after a few months whether cells were initially cultured into media with various corticosterone levels or initially into high levels of corticosterone for 3 weeks prior to the shift to various lower doses.

Hormonal induction of the lactose synthetase components, alpha-lactalbumin and galactosyltransferase, in relation to the induced levels of lactose synthetase activity and lactose secretion by mammary gland explants from mature virgin mice was examined. During 6 days of culture in the presence of insulin, cortisol, prolactin and triiodothyronine mammary explants accumulated progressively increasing amounts of alpha-lactalbumin. By contrast, galactosyltransferase and lactose synthetase activities were maximal on day 3, and then declined; the time course of lactose secretion was similar to that of galactosyltransferase and lactose synthetase activities. Cortisol concentration studies revealed the following dependencies on the steroid: alpha-lactalbumin content was maximal between 0.01 and 0.1 microgram and, at 10 microgram/ml fell below the baseline level observed without cortisol. On the other hand, galactosyltransferase, lactose synthetase and lactose secretion were only slightly increased at 0.01 microgram/ml, were maximal between 0.1 and 1.0 microgram/ml, and at 10 microgram/ml were still considerably above the baseline levels observed without cortisol. These results indicate that lactose formation and secretion by mammary explants from virgin mice correspond more closely to the hormone-induced activity of galactosyltransferase than to that of alpha-lactalbumin, and that asynchrony can arise between the induction of alpha-lactalbumin and the induction of lactose.

The minimal hormonal requirements for the in-vitro accumulation of alpha-lactalbumin have been investigated in a marsupial, the tammar (Macropus eugenii). Mammary gland explants from 24-day pregnant tammars cultured in medium containing bovine insulin, cortisol and ovine prolactin showed a progressive increase in accumulation of alpha-lactalbumin during 4 days of incubation. No increment was observed if prolactin was omitted from the medium. However, a similar rate of increase was observed after 3 days of culture in medium containing prolactin alone. This induction of alpha-lactalbumin was maximal at a prolactin concentration of approximately 0.02 mg/l, which corresponds to physiological levels during pregnancy and early lactation. The absence of an effect of bovine insulin on tammar explants is not due to a general unresponsiveness to this hormone since insulin-stimulated DNA synthesis and amino acid uptake was evident after 3 days of culture. The inclusion of tri-iodothyronine and raised concentrations of cortisol in culture media have been shown to modulate alpha-lactalbumin synthesis in eutherian mammals but were without effect in the tammar. In addition, increased levels of progesterone did not inhibit the induction of alpha-lactalbumin, confirming an earlier in-vivo study suggesting that progesterone withdrawal may not be the lactogenic trigger in this species. Thus the pregnant tammar is the only species yet described in which alpha-lactalbumin is induced maximally in vitro in response to a single hormone.

The content of alpha-lactalbumin and three species of caseins, 42K, 29K, and 25K, have been measured along with the levels and activities of their mRNAs in the rat mammary gland. Changes in these values were followed during gestation and lactation. An increment of 3- to 4-fold over the virgin level was observed for both alpha-lactalbumin and 42K casein during the 1st day of gestation. From this point on, the level of 42K remained unchanged during the 1st week of gestation and increased thereafter. After the increment of the 1st day, the alpha-lactalbumin content decreased rapidly during the 2nd day of gestation, continued to decrease more slowly until the 12th day, and then started to increase thereafter. During the 2nd and 3rd week of gestation. the amounts of alpha-lactalbumin within the gland increased continuously but not uniformly and caseins accumulated rapidly with a tendency to plateau around the 13th to 16th day of gestation. The relative proportions remained, respectively, 42K greater than 29K greater than 25K greater than alpha-lactalbumin until parturition. At the onset of lactation, both alpha-lactalbumin and casein content increased sharply, the relative proportion for caseins changed to 42K greater than 25K greater than 29K greater than alpha-lactalbumin and remained so throughout the lactation period. alpha-Lactalbumin and casein mRNA activity, as judged by the wheat germ translational system, remained unchanged during the 1st week of gestation, then showed a steady but not uniform increase from the 7th day of gestation until parturition. These activities increased sequentially during lactation, alpha-lactalbumin reaching a plateau by the 1st week, caseins between the 1st and 2nd week, and other mRNAs by the end of the 2nd week of lactation. By the 21st day of lactation, the activity of all mRNA had declined. The levels of alpha-lactalbumin mRNA and 16 S doublet casein mRNA sequences measured with the cDNA probes increased by about 8-fold for alpha-lactalbumin mRNA and 6-fold for casein mRNA during the 1st week of gestation. These levels declined slightly early in the 2nd week and then continued to increase until parturition with a shoulder in the levels around the 13th to 16th day. During lactation, these levels increased until the 8th to 12th day and from then on declined. The content of alpha-lactalbumin and caseins, as well as the measurement of sequences and activities of their mRNAs, showed that in the rat mammary gland these differentiated functions are already expressed at the onset of gestation. Both concentration and activity of mRNA are out of phase with protein levels during the 1st week of gestation but they remain in phase thereafter.

Cortisol was previously shown to exert different, concentration-dependent, effects on the accumulation of casein and alpha-lactalbumin in mammary glands from mid-pregnant mice cultured in the presence of insulin and prolactin [Ono & Oka (1980) Cell 19, 473-480]. The present study demonstrated that the addition of 30nM-cortisol to the medium containing insulin and prolactin resulted in a marked enhancement of the rate of synthesis of both alpha-lactalbumin and casein in cultured tissue. The addition of 3 microM-cortisol in combination with insulin and prolactin caused a marked decrease in the rate of alpha-lactalbumin synthesis, but increased casein synthesis substantially. Similar changes were also observed in the amount of translatable mRNA for alpha-lactalbumin and casein in mammary explants cultured with insulin, prolactin and the two concentrations of cortisol. The study of the turnover of the milk proteins in cultured explants showed that virtually all of the casein synthesized remained intact in tissue explants cultured with 3 microM cortisol, whereas about 45% of casein disappeared in 40h from explants cultured with 30nM-cortisol. In contrast, the two concentrations of cortisol did not differentially affect the disappearance of alpha-lactalbumin, which was about 55% in 40h. These results indicate that the concentration-dependent differential actions of cortisol on the accumulation of alpha-lactalbumin and casein are exerted through its effects on the rate of synthesis and turnover of the two proteins as well as on the accumulation of their mRNA species.

The interplay of insulin, cortisol, and prolactin induces synthesis of casein and alpha-lactalbumin in cultured mammary explants from mature virgin mice. A striking difference has been found between the optimal concentrations of cortisol required for maximal induction of the two milk proteins in vitro: 3 x 10(-8) molar for alpha-lactalbumin and 3 x 10(-6) molar for casein. Moreover, 10(-7) to 10(-5) molar cortisol caused progressive inhibition of alpha-lactalbumin accumulation. Such differential actions of cortisol may partly account for the asynchronous synthesis of the two proteins during pregnancy.