Expression of the whey acidic protein in transgenic pigs impairs mammary development

The whey acidic protein has been found in milk of mice, rats, rabbits and camels, and its gene is expressed specifically in mammary tissue at late pregnancy and throughout lactation. A characteristic of whey acidic protein is the ‘four-disulfide-core’ signature which is also present in proteins involved in organ development. We have generated six lines of transgenic pigs which carry a mouse whey acidic protein transgene and express it at high levels in their mammary glands. Transgenic sows from three lines could not produce sufficient quantities of milk to support normal development of healthy offspring. This phenotype appears to be similar, if not identical, to themilchlos phenotype exhibited by mice expressing whey acidic protein transgenes. Mammary tissue from post-partummilchlos sows had an immature histological appearance, which was distinct from that observed during normal development or involution. Expression of the whey acidic protein transgene was found in mammary tissue from sexually immature pigs frommilchlos lines, but not in sows from lines that appeared to lactate normally. We suggest that precocious synthesis of whey acidic protein impairs mammary development and function. Impaired mammary development due to inappropriate timing of whey acidic protein expression is consistent with the notion that proteins with the ‘four-disulfide-core’ signature participate in tissue formation.     

Expression of the whey acidic protein (Wap) is necessary for adequate nourishment of the offspring but not functional differentiation of mammary epithelial cells

Whey acidic protein (WAP) is the principal whey protein found in rodent milk, which contains a cysteine-rich motif identified in some protease inhibitors and proteins involved in tissue modeling. The expression of the Wap gene, which is principally restricted to the mammary gland, increases more than 1,000-fold around mid-pregnancy. To determine whether the expression of this major milk protein gene is a prerequisite for functional differentiation of mammary epithelial cells, we generated conventional knockout mice lacking two alleles of the Wap gene. Wap-deficient females gave birth to normal litter sizes and, initially, produced enough milk to sustain the offspring. The histological analysis of postpartum mammary glands from knockout dams does not reveal striking phenotypic abnormalities. This suggests that the expression of the Wap gene is not required for alveolar specification and functional differentiation. In addition, we found that Wap is dispensable as a protease inhibitor to maintain the stability of secretory proteins in the milk. Nevertheless, a significant number of litters thrived poorly on Wap-deficient dams, in particular during the second half of lactation. This observation suggests that Wap may be essential for the adequate nourishment of the growing young, which triple in size within the first 10 days of lactation. Important implications of these findings for the use of Wap as a marker for advanced differentiation of mammary epithelial cells and the biology of pluripotent progenitors are discussed in the final section.     

Production of human surfactant protein C in milk of transgenic mice

Respiratory distress syndrome (RDS), caused by lack of pulmonary surfactant, affects 65 000 infants annually in the USA. Surfactant replacement therapy reduces the morbidity and mortality associated with RDS. Human surfactant protein C (SP-C) is an important component of pulmonary surfactant. To produce human SP-C, a construct using the rat whey acidic protein (WAP) promoter and 3 untranslated regions to target expression of the human SP-C gene to the mammary gland of transgenic mice was created. WAP/SP-C mRNA expression was detected in all transgenic lines analysed. SP-C was expressed in a copy-number-dependent and integration-site-independent fashion, with levels of expression ranging from 0.01% to 36.0% of the endogenous mouse WAP mRNA, and WAP/SP-C mRNA expresison levels were greater than those of the endogenous mouse lung SP-C mRNA. Expression at the RNA level was specific to the mammary gland and paralleled the endogenous WAP expression pattern during mammary gland development. Expression and secretion of the SP-C protein in the lactating mammary gland was demonstrated by western blots performed on whole milk using an anti-SP-C polyclonal antibody. Immunoreactive proteins of MW 22 and 12–14 kDa appeared only in transgenic milk. The 22 kDa protein represents the proprotein, and the 12–14 kDa is a processed form of SP-C.     

Position-independent and tissue-specific expression of porcine whey acidic protein gene from a bacterial artificial chromosome in transgenic mice

Silencing of transgenes is a frequent event after the random integration of foreign DNA in the host genome following microinjection. Long genomic fragments are expected to contain all the regulatory elements necessary to induce an appropriate expression of transgenes. A bacterial artificial chromosome containing the porcine wap gene with approximately 145 and 5 kb of 5'- and 3'-flanking sequences, respectively, was microinjected into fertilized mouse ovocytes. In the six transgenic lines studied, expression was strictly specific to the mammary gland of lactating animals and was position-independent. Levels of exogenous porcine wap mRNA per copy compared favorably with the porcine wap mRNA yield in the mammary gland of a 9-day lactating pig. These findings suggest that this insert contained most if not all of the cis-acting elements involved in the full specific expression of the porcine wap gene. These elements constitute good candidates for directing the optimized expression of protein recombinant-encoding genes in the mammary gland of lactating animals.     

乳腺生物反应器的产业化进展与展望

进入21世纪,伴随着转基因技术的快速发展,转基因动物的研究已经从方法学研究步入了应用性研究阶段。外源基因在转基因动物的特异性表达,尤其是在乳腺的表达,可将转基因动物用作生物反应器进行生物活性蛋白的生产而应用于商业生产。生产出来的药用蛋白具有纯化简单,投资少,成本低,对环境没有任何污染等优点。用转基因动物生产药用蛋白可获得巨大的经济利益,因此成为国内外研究的热点。     

Quantitative collection of milk and active recombinant proteins from the mammary glands of transgenic mice

Summary

Transgenic mice expressing foreign genes specifically in their mammary glands have been obtained by several groups in the world. The mouse is generally considered as a good reference animal to evaluate the efficiency of gene constructs to be used in larger mammals for the preparation of the corresponding recombinant proteins at an industrial scale. The method described here shows that mammary glands from lactating mice separated from their pups for one day spontaneously released 1.5 ml milk when stored at O'C. The proteins of milk obtained by this method were essentially similar to those obtained after milking. Human growth hormone (hGH) gene under the control of the rabbit whey acidic (WAP) gene promoter was expressed at a high level in the milk of transgenic mice (4 mg/ml milk in the mice examined here). hGH was present in milk obtained after milking or after the incubation of the mammary glands at O'C. In both cases, the hormone was present in essentially similar concentration, undegraded and biologically active (as judged by its prolactin‐like activity). The method depicted here is very simple and can be applied easily to many mice. Its major limitation is that it implies the breeding and the sacrifice of a relatively large number of animals. One gram of crude recombinant protein can be virtually obtained in this way with about 200 lactating mice from their milk containing the proteins at the concentration of 3‐4 mg/ml. The milk of transgenic mice can therefore be considered as a practical source of recombinant proteins for biochemical and pharmaceutical studies.     

利用转基因植物生产药用蛋白研究进展

简要评述了国内外利用转基因植物生产药用蛋白的研究现状、发展趋势,以及转基因植物生产药用蛋白的基本方法、应用研究等。尽管目前植物作为药用蛋白的生物反应器受到诸多因素限制,优点与问题并存,但利用转基因植物生产药用蛋白是植物基因工程研究领域的一个新的发展趋势。     

Recombinant human bile salt-stimulated lipase: an example of defectiveO-glycosylation of a protein produced in milk of transgenic mice

The expression of recombinant human bile salt-stimulated lipase (bssl) was targeted to the lactating mammary gland of transgenic mice. Expression of recombinant genes comprisingbssl cDNA, or alternatively genomicbssl DNA, under control of regulatory elements derived from the murine whey acidic protein (wap) gene was achieved and evaluated. Constructs containing genomicbssl sequences mediated high levels (0.5–1, mg ml^–1) of recombinant human BSSL in the milk. The recombinant BSSL produced was purified, biochemically characterized and compared to native BSSL and recombinant BSSL produced in mouse C127 and hamster CHO cells. Recombinant BSSL derived from transgenic mice showed a different migration and distribution after SDS-PAGE electrophoresis, lower apparent molecular mass on size-exclusion chromatography and no detectable interactions with a panel of lectins. These results indicate a significantly lower degree ofO-glycosylation of recombinant BSSL in milk from transgenic mice than was found for the native enzyme or recombinant CHO- or C127 cell-produced BSSL. Despite these differences, mouse-milk-derived recombinant BSSL exhibited similar lipase activity, the same, stability to low pH and similar sensitivity to elevated temperatures as the native enzyme. The observation that mouse-C127-cell-produced recombinant BSSL is heavilyO-glycosylated makes species-related restrictions less attractive as an explanation for the reducedO-glycosylation.     

Phenotypic and genotypic stability of multiple lines of transgenic pigs expressing recombinant human protein C

The genotypic and phenotypic stability of four lines of transgenic pigs expressing recombinant human protein C in milk was examined. Two lines were established with a construct consisting of a 2.6 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Two lines were established with another construct consisting of a 4.1 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Genotypic stability was measured by transgene copy number transmission. Outbred offspring having a single transgene integration locus were established from a founder having three independent, multicopy loci. Phenotypic stability over multiple lactations was defined by the combination of recombinant human protein C expression levels and the isoform signature of recombinant human protein C in western blots. Both cDNA and genomic human protein C transgenes gave similar ranges of expression levels of about 100--1800 g ml–1. Within a given outbred lineage having a single loci for the cDNA transgene, the expression levels ranged between 100--400 g ml–1. Western blots of reduced recombinant protein C revealed that single chain content was not dependent on expression level and was consistent within each transgenic line, but varied between transgenic lines. This suggests that native swine genetics may play a role in selection of production herds with optimal post-translational proteolytic processing capability. Although swine are not conventional dairy livestock, it is agreed that the short generation times, multiple offspring per litter, stable paternal transmission of the transgene, and milk production capabilities of swine offer distinct advantages over conventional dairy livestock for the establishment of a herd producing a therapeutic recombinant protein     

Expression of human lysozyme mRNA in the mammary gland of transgenic mice

Owing to its inherent antimicrobial effect and positive charge, the expression of human lysozyme in bovine milk could be beneficial by altering the overal microbial level and the functional and physical properties of the milk. We have used transgenic mice as model systems to evaluate the expression of human lysozyme containing fusion gene constructs in the mammary gland. Expression of human lysozyme was targeted to the mammary gland by using the 5 promoter elements of either the bovine (line B mice) or _s1 (line H mice) casein genes coupled to the cDNA for human lysozyme. Expression of human lysozyme mRNA was not found in mammary tissue from any of line B mice. Tissues were analysed from six lines of H mice and two, H6 and H5, were found to express human lysozyme mRNA in the mammary gland at 42% and 116%, respectively, of the levels of the endogenous mouse whey acidic protein gene. At peak lactation, female mice homozygous for the H5 and H6 transgene have approximately twice the amount of mRNA encoding human lysozyme as hemizygous animals. Expression levels of human lysozyme mRNA in the mammary gland at time points representing late pregnancy, early, peak and late lactation corresponded to the profile of casein gene expression. Human lysozyme mRNA expression was not observed in transgenic males, virgin females or in the kidney, liver, spleen or brain of lactating females. A very low level of expression of human lysozyme mRNA was observed in the salivary gland of line H5.     

Targeting the exogenous htPAm gene on goat somatic cell beta-casein locus for transgenic goat production

Combining gene targeting of animal somatic cells with nuclear transfer technique has provided a powerful method to produce transgenic animal mammary gland bioreactor. The objective of this study is to make an efficient and reproducible gene targeting in goat fetal fibroblasts by inserting the exogenous htPAm cDNA into the beta-casein locus with liposomes or electroporation so that htPAm protein might be produced in gene-targeted goat mammary gland. By gene-targeting technique, the exogenous htPAm gene was inserted to milk goat beta-casein gene sequences. Fetal fibroblasts were isolated from Day 35 fetuses of Guanzhong milk goats, and transfected with linear gene-targeting vector pGBC4htPAm using Lipefectamin-2000 and electoporation, respectively. Forty-eight gene-targeted cell colonies with homologous recombination were obtained, and three cell colonies were verified by DNA sequence analysis within the homologous recombination region. Using gene-targeted cell lines as donor cells for nuclear transfer, a total of 600 reconstructed embryos had been obtained, and 146 developed cloned embryos were transferred to 16 recipient goats, and finally three goats showed pregnancy at Day 90.     

体细胞基因打靶制备动物乳腺生物反应器的策略与应用

在转基因动物研究中,由于基因表达调控元件的人工拼接和外源基因在动物基因组中随机整合所带来的“位置效应”,致使转基因动物外源基因的表达水平不高并且差异较大。为此,利用定位整合优势,对以基因同源重组为基础的基因打靶技术进行了大量研究。介绍了就利用体细胞基因打靶和核移植技术制备动物乳腺生物反应器的策略和应用情况做一综述,并对提高基因打靶效率的各种策略,打靶细胞的选择,转基因细胞核移植的低融合事件以及基因打靶制备乳腺生物反应器的优越性进行分析。     

Synthesis and secretion of caseins by the mouse mammary gland: Production and characterization of new polyclonal antibodies

Polyclonal antibodies to mouse - and /-caseins were raised in rabbits. These antibodies display tissue- and species specificity as shown by immunoblotting. Immunohistochemical analyses demonstrate that both - and /-caseins were synthesized and secreted from virtually all lactating mammary epithelial cells, in a pattern very similar to that of the mouse -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 -casein in the milk, the amount of this protein compared to - or -caseins was extremely low in medium conditioned for 24 h by mammary explants of mid-pregnant mice immediately after explantation or after 4 days.