Production of transgenic dairy goat expressing human α-lactalbumin by somatic cell nuclear transfer.

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 ß-lactoglobulin (ßLG) 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.

GWA analysis for milk production traits in dairy sheep and genetic support for a QTN influencing milk protein percentage in the LALBA gene.

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.

Formula feeding skews immune cell composition toward adaptive immunity compared to breastfeeding.

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.

Characterizing transgene inheritance.

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.

Expression of genes involved in regulation of cell turnover during milk stasis and lactation rescue in sow mammary glands.

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.

Transfer of an expression YAC into goat fetal fibroblasts by cell fusion for mammary gland bioreactor.

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.

Association of increased estrogen receptor beta2 expression with parity-induced alterations in the rat mammary gland.

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.

The mechanisms by which nitric oxide affects mammary epithelial growth and differentiation.

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.

Short communication: effects of increased expression of alpha-lactalbumin in transgenic mice on milk yield and pup growth.

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.

Improvements in human health through production of human milk proteins in transgenic food plants.

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.

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.

Production and breeding of transgenic cattle using in vitro embryo production technology.

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.

Remarkable destabilization of recombinant alpha-lactalbumin by an extraneous N-terminal methionyl residue.

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.

Production of bovine alpha-lactalbumin in the milk of transgenic pigs.

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.

Position-independent and high-level expression of human alpha-lactalbumin in the milk of transgenic rats carrying a 210-kb YAC DNA.

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.

Bovine mammary explant versus primary cell cultures: effect of bovine somatotropin and insulin-like growth factor-I on DNA content and protein synthesis.

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.

Effect of prolactin on in vitro expression of the bovine mammary immunoglobulin G1 receptor.

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.

Characterization and partial purification of bovine alpha-lactalbumin and beta-casein produced in milk of transgenic mice.

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.

Production of proteins in the milk of transgenic livestock: problems, solutions, and successes.

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.

Estrogen administered at final milk removal accelerates involution of bovine mammary gland.

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.