Isolation and some effects of functional, low-phenylalanine kappa-casein expressed in the milk of transgenic rabbits

Patients suffering certain metabolic diseases (e.g. phenylketonuria) need a low-phenylalanine diet throughout their lives. Transgenic rabbits were created to express low-phenylalanine kappa-casein in their milk. The aim was to demonstrate for the first time the feasibility of producing a modified milk protein in addition to normal milk proteins. A gene construct containing the coding region of the rabbit kappa-casein gene was modified by site-specific oligonucleotide directed mutagenesis. Four of the five phenylalanine amino acids present in the mature protein were mutated and the gene construct was used to create two transgenic rabbit lines. The transgenic rabbits produced the recombinant kappa-casein at a high level in their milk causing a reduction in the average size of the casein micelles. The low-phenylalanine kappa-casein was digestible with chymosin and it was separated from its native counterpart and from the other milk proteins by a one-step HPLC method on a reversed-phase column. In the future, low-phenylalanine casein produced in transgenic animals could be used as dietary replacements to meet the special requirements of certain consumer groups.     

Compositional analysis of dairy products derived from clones and cloned transgenic cattle

Cloning technology is an emerging biotechnological tool that could provide commercial opportunities for livestock agriculture. However, the process is very inefficient and the molecular events underlying the technology are poorly understood. The resulting uncertainties are causing concerns regarding the safety of food products derived from cloned livestock. There are similar concerns for livestock produced by biotechnologies which enable the purposeful introduction of genetic modifications. To increase the knowledge about food products from animals generated by these modern biotechnologies, we assessed compositional differences associated with milk and cheese derived from cloned and transgenic cows. Based on gross composition, fatty acid and amino acid profiles and mineral and vitamin contents, milk produced by clones and conventional cattle were essentially similar and consistent with reference values from dairy cows farmed in the same region under similar conditions. Whereas colostrum produced by transgenic cows with additional casein genes had similar IgG secretion levels and kinetics to control cows, milk from the transgenic cows had a distinct yellow appearance, in contrast to the white color of milk from control cows. Processing of milk into cheese resulted in differences in the gross composition and amino acid profiles; 'transgenic' cheese had lower fat and higher salt contents and small but characteristic differences in the amino acid profile compared to control cheese.     

Associations between milk performance traits in Holstein cows and 16 candidate SNPs identified by arrayed primer extension (APEX) microarray

An oligonucleotide microarray-which allows for parallel genotyping of many SNPs in genes involved in cow milk protein biosynthesis-was used to identify which of the 16 candidate SNPs are associated with milk performance traits in Holstein cows. Four hundred cows were genotyped by the developed and validated microarray. Significant associations were found between four single SNPs, namely DGAT1 (acyloCoA:diacylglycerol acyltransferase), LTF (lactoferrin), CSN3 (kappa-casein), and GHR (growth hormone receptor) and with fat and protein yield and percentage. Many significant associations between combined genotypes (two SNPs) and milk performance traits were found. The associations between the combined genotypes DGAT1/LTF and DGAT1/LEPTIN analyzed traits are presented as examples. The microarray based on APEX (Arrayed Primer Extension) is a fast and reliable method for multiple SNP analysis of potential application in marker-assisted selection. After further development, the chip may prospectively be used for dairy cattle paternity analysis and evolutionary studies.     

Associations Between Milk Performance Traits in Holstein Cows and 16 Candidate SNPs Identified by Arrayed Primer Extension (APEX) Microarray

An oligonucleotide microarray—which allows for parallel genotyping of many SNPs in genes involved in cow milk protein biosynthesis—was used to identify which of the 16 candidate SNPs are associated with milk performance traits in Holstein cows. Four hundred cows were genotyped by the developed and validated microarray. Significant associations were found between four single SNPs, namely DGAT1 (acyloCoA:diacylglycerol acyltransferase), LTF (lactoferrin), CSN3 (kappa-casein), and GHR (growth hormone receptor) and with fat and protein yield and percentage. Many significant associations between combined genotypes (two SNPs) and milk performance traits were found. The associations between the combined genotypes DGAT1/LTF and DGAT1/LEPTIN analyzed traits are presented as examples.

The microarray based on APEX (Arrayed Primer Extension) is a fast and reliable method for multiple SNP analysis of potential application in marker-assisted selection. After further development, the chip may prospectively be used for dairy cattle paternity analysis and evolutionary studies.     

The influence of extracellular matrix and prolactin on global gene expression profiles of primary bovine mammary epithelial cells in vitro

An in vitro bovine mammosphere model was characterized for use in lactational biology studies using a functional genomics approach. Primary bovine mammary epithelial cells cultured on a basement membrane, Matrigel, formed three-dimensional alveoli-like structures or mammospheres. Gene expression profiling during mammosphere formation by high-density microarray analysis indicated that mammospheres underwent similar molecular and cellular processes to developing alveoli in the mammary gland. Gene expression profiles indicated that genes involved in milk protein and fat biosynthesis were expressed, however, lactose biosynthesis may have been compromised. Investigation of factors influencing mammosphere formation revealed that extracellular matrix (ECM) was responsible for the initiation of this process and that prolactin (Prl) was necessary for high levels of milk protein expression. CSN3 (encoding κ-casein) was the most highly expressed casein gene, followed by CSN1S1 (encoding αS1-casein) and CSN2 (encoding β-casein). Eighteen Prl-responsive genes were identified, including CSN1S1 , SOCS2 and CSN2, however, expression of CSN3 was not significantly increased by Prl and CSN1S2 was not expressed at detectable levels in mammospheres. A number of novel Prl responsive genes were identified, including ECM components and genes involved in differentiation and apoptosis. This mammosphere model is a useful model system for functional genomics studies of certain aspects of dairy cattle lactation.     

Comparison of casein of cynomolgus monkey (Macaca fascicularis) with human casein

Casein of cynomolgus monkey was compared with those from human and bovine milk. Cynomolgus monkey casein showed similar electrophoretical patterns to those of human casein on Disc- and SDS-electrophoresis. It consisted of beta- and kappa-casein-like components. The component corresponding to bovine alpha s1-casein was not detected. The beta-casein-like fraction of cynomolgus monkey showed 9 bands on Disc-PAGE. These were suggested to be the same protein binding different levels of phosphorus by dephosphorylation experiment using an acid phosphatase. The kappa-casein-like component of cynomolgus monkey was highly glycosylated (about 50% carbohydrate) similarly as human kappa-casein and the constituent carbohydrates were same as those detected in human kappa-casein (galactose, fucose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acid). Amino acid composition of cynomolgus monkey kappa-casein bore a resemblance to those of both human and bovine kappa-caseins. Amino acid composition of cynomolgus monkey beta-casein was also similar to those of human and bovine beta-caseins.     

Identification of human milk kappa-casein on polyacrylamide gels by differential staining with Ethyl-Stains-all and chymosin sensitivity

Ethyl-Stains-all (ESA), a cationic carbocyanine dye that stains phosphorylated, sialylated, and unmodified proteins differentially, was used to stain a human casein fraction enriched for its kappa-casein-like characteristics. The staining properties and chymosin sensitivity of this fraction were compared with those of human milk and bovine casein proteins. Phosphorylated human and bovine beta caseins stained blue with ESA. The sialic acid-containing bovine kappa-casein stained blue-green. The human kappa-like fraction was enriched for a protein that stained blue-green with ESA. Both bovine kappa-casein and the human blue-green-staining protein were susceptible to chymosin digestion at lower concentrations of chymosin than that required for digestion of beta-caseins. In each case, following chymosin digestion, a green-staining peptide of lower molecular weight replaced the original protein and para-kappa-casein was formed. Identification of human kappa-casein on SDS-polyacrylamide gels was based on its differential staining with ESA and chymosin sensitivity with respect to beta-casein.     

Associations between 2 paternal casein haplotypes and milk yield traits of Swiss Fleckvieh cattle

Associations between casein haplotypes and milk yield traits of offspring from 5 Swiss Fleckvieh AI test bulls were investigated. The analysis was performed by using a daughter design, where each daughter inherited either paternal haplotype B-A1-A-A or B-A2-A-A for alleles of alpha s1-, beta-, alpha s2- and kappa-casein genes. The substitution effects of paternal CSN2 A1 versus A2 on protein yield deviations (YDs) were significant (P < 0.05), whereas their effects on milk and fat YDs were not. The paternal substitution effects of the CSN2 A1 versus the A2 allele on protein YDs within the 5 sires did not reach the significance level. This is due to the contrary allele substitution effect of a sire compared to the other 4 sires. The effects of maternal haplotypes on milk, protein and fat YDs were not significant. However, it is noteworthy that the effects of haplotypes with a low frequency in the population deviate largely from the most frequent haplotype B-A2-A-A. The effects of beta-lactoglobulin (BLG) genotypes were significant for protein YDs but not for milk and fat YDs. The association between the paternal CSN2 A1 and A2 alleles and milk protein YDs within sires but not milk and fat YDs indicate an interaction, which might be a consequence of CSN2 heterogeneity or a closely linked gene that is contributing to the estimated effects.     

A 3,387?bp 5′-flanking sequence of the goat alpha-S1-casein gene provides correct tissue-specific expression of human granulocyte colony-stimulating factor (hG-CSF) in the mammary gland of transgenic mice

A new expression vector containing the 1,944 bp 5'-flanking regulatory region together with exon 1 and intron 1 of the goat alpha-S1-casein gene (CSN1S1), the full-sized human granulocyte colony-stimulating factor gene (hGCSF) and the 3'-flanking sequence of the bovine CSN1S1, was created. The vector DNA was used for generation of four mouse transgenic lines. The transgene was integrated into chromosomes 8 and 12 of two founders as 2 and 5 copies, respectively. Tissue-specific secretion of hG-CSF into the milk of transgenic mice was in the range of 19-40 μg/ml. RT-PCR analysis of various tissues of the transgenic mice demonstrated that expression of hGCSF was detected in only the mammary gland in the progeny of all founders. Moreover, cells were shown to be positive for hG-CSF by immunofluorescent analysis in the mammary glands but not in any other tissues. There were no signs of mosaic expression in the mammary gland. Trace amounts of hG-CSF were detected in the serum of females of two transgenic lines during lactation only. However, no transgenic mice showed any changes in hematopoiesis based on the number of granulocytes in blood. Immunoblotting of hG-CSF in the milk of transgenic mice revealed two forms, presumably the glycosylated and non-glycosylated forms. The hematopoietic activity of hG-CSF in the milk of transgenic females is comparable to that of recombinant G-CSF. In general, the data obtained in this study show that the new expression vector is able to provide correct tissue-specific expression of hG-CSF with high biological activity in transgenic mice.     

The identification of the kappa-casein genotype in Holstein dairy cattle using the polymerase chain reaction

Summary The polymerase chain reaction (PCR) was used to amplify a 99-bp region from the kappa-casein gene of Holstein dairy cattle which contains nucleotide substitutions that are diagnostic of the two major protein variants of kappa-casein. Identity of the amplified product was confirmed by direct sequencing. Digestion of the PCR product with MboII (A-variant specific) or TaqI (B-variant specific) allowed direct determination of the genotype of the animal (homozygous or heterozygous). A total of 58 lactating cows with known kappa-casein phenotype were tested using PCR. In all cases, the measured genotype confirmed the phenotype. We have also tested the genotype of 42 sires that were top ranked for milk yield by the CIAQ (Centre d'insemination artificielle du Quebec). The B-allele of kappa-casein which occurred at a frequency of 0.13 among the proven bulls is associated with superior milk for industrial applications. Identification of the kappa-casein genotype by PCR in bulls and calves would provide a means for rapidly changing the frequency of the B-allele in the breeding population by selection.     

A PCR-RFLP test to detect allelic variants of the bovine kappa-casein gene

Point mutations in exon IV of bovine kappa-casein gene (kappaCn, CASK, CSN3) determine nine allelic variants (A, B, C, E, F, G, H, I, and A1) for the gene. These variants are associated with major differences in composition and manufacturing properties of milk (i.e., cheese yield). A PCR-RFLP test was developed in order to distinguish the different alleles. Polymorphisms are detected by digestion with the endonucleases HindIII, HaeIII, and MaeII followed by electrophoresis in agarose gels stained with ethidium bromide. Twenty eight DNA samples from different breeds of Argentina were analyzed for the A, B, and E variants. This simple PCR-RFLP test makes feasible the inclusion of kappa-casein genotypes in breeding plans.     

Suitability of animals' purified milk caseins and their subunit kappa-caseins as substrates for subtilisin and trypsin.

Acid casein and kappa-casein were purified from different species of animal's milk, such as cow, sheep, goat, and water buffalo. These caseins were used as substrates for commercially available subtilisin and trypsin. It was established that, when acid caseins were used as a substrate for subtilisin, cow acid casein was found to be a better substrate for the enzymes, compared to other animals' milk casein. It was suggested that this acid casein has significantly more aromatic amino acids, as compared to arginine and lysine. K(M) and Vmax values, which were obtained for cow kappa-casein, showed that cow kappa-casein was a better susbstrate for trypsin than the others, suggesting that cow kappa-casein has a rich content of lysine, arginine, and aromatic amino acids by comparison with the others. The calculated C/N ratio also supports this suggestion.     

Mouse mammary tumor virus promoter directs high‐level expression of bovine αS1 casein in the milk of transgenic heterozygous and homozygous mice

Abstract

To test the mouse mammary tumor virus (MMTV) promoter as an inducible mammary‐specific promoter, we have produced 3 lines of transgenic mice carrying bovine αS1 casein cDNA under the control of MMTV promoter. The RNA analysis showed that in line 27, heterozygotes expressed 25%, and homozygotes 37% of the endogenous αSI casein mRNA of a mid‐lactation cow. Dexamethasone increased expression by about 3 fold in both heterozygotes and homozygotes. A similar increase in the level of mRNA was observed in both heterozygotes and homozygotes from line 42 with/without induction by dexamethasone. The transgenes were expressed predominantly in the mammary gland with low levels in the salivary gland, spleen, lung, and kidney. Their expression in mammary glands was lactation‐specific. The offspring from lines 27 and 42 expressed a high‐level bovine αS1 casein in their milk. Their expression in milk were 0.21 and 0.11 g/L for heterozygotes, 0.36 and 0.19 g/L for homozygotes, respectively. Dexamethasone further increased the levels of expression by approximately two fold for both heterozygotes and homozygotes.     

A comprehensive study of the relationship between size and protein composition in natural bovine casein micelles

Casein micelles of bovine skimmed milk were fractionated by permeation chromatography on porous glass (CPG-10, 50 nm followed by CPG-10, 300 nm) at 30 degrees C. Micelles were pooled in eight eluant fractions and their size distribution was determined by electron microscopy. The composition of casein in the eight fractions was determined by quantitative hydroxyapatite chromatography. Micelle size decreased progressively with increasing elution volume, and volume-to-surface average diameter ranged from 154 nm in fraction 1 to 62 nm in fraction 8. Concurrently there was a decrease in relative proportions of alpha s- and beta-caseins and a large enrichment of kappa-casein, which changed from 4.1% total casein in fraction 1 to 12.1% total casein in fraction 8. At least half the decrease in alpha s-casein proportions was attributed to the alpha s1-casein component, but the data also suggested a decline in proportions of alpha s2-casein in the smallest micelle fractions. A plot of kappa-casein fractional content versus micelle surface-to-volume ratio gave a straight line (correlation coefficient from linear regression 0.98) from which an average kappa-casein surface coverage of 1.5 m2/mg or 47.3 nm2/molecule was obtained. If a constant surface coverage for kappa-casein is assumed, the parameters of the linear equation predict that micelle voluminosity is inversely related to micelle diameter, being approximately 30% larger in fraction 8 compared to fraction 1.     

Casein α s1 is expressed by human monocytes and upregulates the production of GM-CSF via p38 MAPK

Caseins are major constituents of mammalian milks that are thought to be exclusively expressed in mammary glands and to function primarily as a protein source, as well as to ameliorate intestinal calcium uptake. In addition, proinflammatory and immunomodulatory properties have been reported for bovine caseins. Our aim was to investigate whether human casein α s1 (CSN1S1) is expressed outside the mammary gland and possesses immunomodulatory functions in humans as well. For this purpose, CSN1S1 mRNA was detected in primary human monocytes and CD4(+) and CD8(+) T cells, but not in CD19(+) B cells. CSN1S1 protein was traceable in supernatants of cultured primary human CD14(+) monocytes by ELISA. Similarly, CSN1S1 mRNA and protein were detected in the human monocytic cell lines HL60, U937, and THP1 but not in Mono Mac 6 cells. Moreover, permeabilized human monocytes and HL60 cells could be stained by immunofluorescence, indicating intracellular expression. Recombinant human CSN1S1 was bound to the surface of Mono Mac 6 cells and upregulated the expression of GM-CSF mRNA in primary human monocytes and Mono Mac 6 cells in a time- and concentration-dependent manner. A similar increase in GM-CSF protein was found in the culture supernatants. CSN1S1-dependent upregulation of GM-CSF was specifically blocked by the addition of the p38 MAPK inhibitor ML3403. Our results indicated that human CSN1S1 may possess an immunomodulatory role beyond its nutritional function in milk. It is expressed in human monocytes and stimulates the expression of the proinflammatory cytokine GM-CSF.     

Amyloid fibril formation by bovine milk kappa-casein and its inhibition by the molecular chaperones alphaS- and beta-casein

Caseins are a unique and diverse group of proteins present in bovine milk. While their function is presumed to be primarily nutritional, caseins have a remarkable ability to stabilize proteins, i.e., to inhibit protein aggregation and precipitation, that is comparable to molecular chaperones of the small heat-shock protein (sHsp) family. Additionally, sHsps have been shown to inhibit the formation of amyloid fibrils. This study investigated (i) the fibril-forming propensities of casein proteins and their mixture, sodium caseinate, and (ii) the ability of caseins to prevent in vitro fibril formation by kappa-casein. Transmission electron microscopy (TEM) and X-ray fiber diffraction data demonstrated that kappa-casein readily forms amyloid fibrils at 37 degrees C particularly following reduction of its disulfide bonds. The time-dependent increase in thioflavin T fluorescence observed for reduced and nonreduced kappa-casein at 37 degrees C was suppressed by stoichiometric amounts of alphaS- and beta-casein and by the hydrophobic dye 8-anilino-1-naphthalene sulfonate; the inhibition of kappa-casein fibril formation under these conditions was verified by TEM. Our findings suggest that alphaS- and beta-casein are potent inhibitors of kappa-casein fibril formation and may prevent large-scale fibril formation in vivo. Casein proteins may therefore play a preventative role in the development of corpora amylacea, a disorder associated with the accumulation of amyloid deposits in mammary tissue.     

Transgene expression in mammary glands of newborn rats

Transgene expression in the mammary glands of newborn rats was studied to establish an early selection system for transgenic animals producing exogenous proteins in their milk during lactation. A fusion gene composed of the bovine alpha S1 casein gene promoter and the human growth hormone gene was microinjected into rat embryos. Transgenic lines that produced human growth hormone in their milk were established and used in this study. Immediately after birth, and without any hormone treatment, human growth hormone was found in the extracts of mammary glands from both male and female rats derived from the line secreting human growth hormone in their milk. The expression of the transgene in mammary glands of newborn rats was also detected by the presence of human growth hormone mRNA. Nontransgenic newborn rats did not express the human growth hormone gene in their mammary glands, while the mRNA for rat alpha casein, an endogenous milk protein, was found in all mammary glands from both transgenic and nontransgenic neonates. These results show that analyzing the expression of transgenes in the mammary glands of neonates is a valuable tool to select the desired transgenic animals and to shorten the selection schedules establishing the transgenic animals. © 1996 Wiley-Liss, Inc.