Adenoviral vector mediates high expression levels of human lactoferrin in the milk of rabbits

limitations in current technology for generating transgenic animals, such as the time and the expense, hampered its extensive use in recombinant protein production for therapeutic purpose. In this report, we present a simple and less expensive alternative by directly infusing a recombinant adenovirus vector carrying human lactoferrin cDNA into rabbit mammary glands. The milk serum was collected from the infected mammary gland 48 h post-infection and subjected to a 10% SDS-PAGE and Western blotting. An 80-kDa protein was visualized after viral vector infection. With this method, we obtained a high level of expressed human lactoferrin of up to 2.3 mg/ml in the milk. Taken together, the method is useful for the transient high-level expression recombinant proteins, and the approach established here is probably one of the most economical and efficient ways for large-scale production of recombinant proteins of biopharmaceutical interest.     

Differential in vitro and in vivo glycosylation of human erythropoietin expressed in adenovirally transduced mouse mammary epithelial cells

The expression of human erythropoietin in the mammary gland is an attractive approach to diminish its current production cost. Previous attempts to produce erythropoietin in the milk of transgenic animals resulted in very low expression levels and in a detrimental effect in the health of the founder animals. Here, we show that the direct transduction of the mouse mammary gland with an adenoviral vector carrying the cDNA of erythropoietin promotes its expression in milk at a level as high as 3.5 mg/ml. The recombinant erythropoietin derived from mouse milk showed a different migration and distribution after SDS-PAGE electrophoresis as well as a low in vivo hematopoietic activity. Enzymatic deglycosylation showed that these molecular weight disparities are in part due to differential glycosylation compared to with its counterpart produced in CHO and HC11 cell lines. The difference between in vivo and in vitro glycosylation of human erythropoietin expressed in adenovirally transduced mammary epithelial cells suggests that key enzymes in the glycosylation pathway may be insufficient during lactation. Thus, the direct transduction of the mammary epithelium seems to be a powerful tool to express toxic proteins in milk at levels high enough for their physical, chemical and biological characterization before undertaking the generation of a transgenic mammal.     

Expression of human growth hormone in the milk of transgenic rabbits with transgene mapped to the telomere region of chromosome 7q

The advent of transgenic technology has provided methods for the production of pharmaceuticals by the isolation of these proteins from transgenic animals. The mammary gland has been focused on as a bioreactor, since milk is easily collected from lactating animals and protein production can be expressed at very high levels, including hormones and enzymes. We demonstrate here the expression pattern of recombinant human growth hormone (rhGH) in transgenic rabbits carrying hGH genomic sequences driven by the rat whey acidic protein (WAP) promoter. The transgene was mapped to the q26-27 telomere region of chromosome 7q by fluorescence in situ hybridization (FISH). Nearly 30 % of the F1 generation demonstrated the presence of transgene. The recombinant growth hormone was detected in the milk of the transgenic rabbit females, but not in serum, up to the level of 10???g/ml. Ectopic expression of the transgene in the brain, heart, kidney, liver, and salivary gland was not observed, indicating that a short sequence of rat WAP promoter (969 bp) contained essential sequences directing expression exclusively to the mammary gland. The biological activity of recombinant growth hormone was measured by immunoreactivity and the capability to stimulate growth of the hormone-dependent Nb211 cell line.     

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.     

High-level expression of human lactoferrin in the milk of goats by using replication-defective adenoviral vectors

The expression of human lactoferrin in the mammary gland is an attractive approach to diminish its current production cost. Previous attempts to produce lactorferrin in the milk of transgenic animals resulted in very high cost and uncertain results. In this paper, we have directly infused replication-defective adenovirus encoding human lactoferrin cDNA into the mammary gland of goats via the teat canal. In this way, we obtained a high level of expressed human lactoferrin up to 2g/L in the milk of goats. The milk serum was collected from the infected mammary gland 48 h post-infection and subjected to a 10% SDS-PAGE and Western blotting. A approximately 80-kDa protein was visualized after viral vector infection. Our results demonstrate that intraductal injection of recombinant replication-defective adenovirus vectors may provide a very useful tool for large-scale production of recombinant proteins of biopharmaceutical interest.     

重组腺病毒介导的人尿激酶原突变体在山羊乳汁中的表达

构建携带人尿激酶原突变体cDNA的重组腺病毒,通过该腺病毒介导实现外源基因在山羊乳腺中表达。通过大肠杆菌内同源重组将人尿激酶原突变体cDNA插入到腺病毒载体中,经过293细胞包装获得重组腺病毒,直接注射到泌乳山羊乳腺,收集感染后1~4天的乳汁,利用纤维蛋白溶圈试验、Western blot、ELISA检测乳清中尿激酶原突变体的表达。结果显示病毒注射后1~4天乳清中均可检测到尿激酶原的表达,其表达量可达0.41mg/ml。该方法可以实现重组蛋白在山羊乳腺的短期表达,可能是大规模生产临床医疗蛋白的一条经济有效的途径。     

人促血小板生成素在转基因小鼠乳腺中定位表达的研究(英文)

通过转基因动物乳腺生物反应器大规模生产药用蛋白质已成为现代生物技术新的生长点之一。为研制表达人促血小板生成素的哺乳动物生物反应器的转基因小鼠模型,本论文以小鼠乳清酸蛋白 (mWAP) 基因5挾说骺厍团-s1-酪蛋白基因3挾说骺厍魑鹘谠菇擞糜诒泶锶舜傺“迳伤氐娜橄僮橹匾煨员泶镌靥錺WAPTPO(Fig.1)。通过常规显微注射的方法把mWAP启动子指导的hTPO表达载体导入小鼠受精卵,获得出生小鼠16只。经PCR检测,有6只为转基因阳性(Fig.2)。G0代小鼠中转基因整合率为37.5% (6/16),用ELISA方法在G0代转基因雌鼠的乳汁中检测了促血小板生成素的表达,表达量在0.8 mg/mL以上(Table 1)。这些结果表明我们已建立了乳腺表达hTPO 的转基因小鼠模型,为以后大型家畜乳腺生物反应器的研制提供了科学依据。     

Generation and Phenotypic Analysis of a Transgenic Line of Rabbits Secreting Active Recombinant Human Erythropoietin in the Milk

Production of recombinant human erythropoietin (rhEPO) for therapeutic purposes relies on its expression in selected clones of transfected mammalian cells. Alternatively, this glycoprotein can be produced by targeted secretion into the body fluid of transgenic mammals. Here, we report on the generation of a transgenic rabbits producing rhEPO in the lactating mammary gland. Transgenic individuals are viable, fertile and transmit the rhEPO gene to the offspring. Northern blot data indicated that the expression of the transgene in the mammary gland is controlled by whey acidic protien (WAP) regulatory sequences during the period of lactation. While the hybridization with total RNA revealed the expression only in the lactating mammary gland, the highly sensitive combinatory approach using RT-PCR/hybridization technique detected a minor ectopic expression. The level of rhEPO secretion in the founder female, measured in the period of lactation, varied in the range of 60–178 and 60–162 mIU/ml in the milk and blood plasma, respectively. Biological activity of the milk rhEPO was confirmed by a standard [³H]-thymidine incorporation test. Thus, we describe the model of a rhEPO-transgenic rabbit, valuable for studies of rhEPO glycosylation and function, which can be useful for the development of transgenic approaches designed for the preparation of recombinant proteins by alternative biopharmaceutical production.     

High expression level of recombinant human erythropoietin in the milk of non-transgenic goats

The high degree of structural conservation of erythropoietin between species, make it, especially, difficult to produce this protein growth factor in the milk of transgenic animals. Here, we show that through the direct transduction of the mammary epithelium, it is possible to produce high levels of recombinant human erythropoietin in the milk of non-transgenic goats without causing harm to the animals. The efficiency of viral transduction was improved through a temporal disruption of tight-junctions with EGTA allowing for the expression of human erythropoietin at levels of up to 2g/L in milk. The human erythropoietin was purified from the milk using a multi-step protocol involving milk clarification, two precipitation steps and two affinity chromatographies, with a yield of about 70% and purity over 98%. However, the human erythropoietin expressed in milk was underglycosylated, which seems to be the main cause for its low in vivo hematopoietic activity. Nonetheless, these results demonstrate that through the direct transduction of the mammary epithelium it is possible to produce potentially toxic proteins in milk, at levels high enough for their purification and biological characterization.     

Large scale production of recombinant human lactoferrin in the milk of transgenic cows

The limited capacity of current bioreactors has led the biopharmaceutical industry to investigate alternative protein expression systems. The milk of transgenic cattle may provide an attractive vehicle for large-scale production of biopharmaceuticals, but there have been no reports on the characteristics of such recombinant proteins. Here we describe the production of recombinant human lactoferrin (rhLF), an iron-binding glycoprotein involved in innate host defense, at gram per liter concentrations in bovine milk. Natural hLF from human milk and rhLF had identical iron-binding and -release properties. Although natural hLF and rhLF underwent differential N-linked glycosylation, they were equally effective in three different in vivo infection models employing immunocompetent and leukocytopenic mice, and showed similar localization at sites of infection. Taken together, the results illustrate the potential of transgenic cattle in the large-scale production of biopharmaceuticals.     

Hybrid expression cassettes consisting of a milk protein promoter and a cytomegalovirus enhancer significantly increase mammary-specific expression of human lactoferrin in transgenic mice

It is very important to develop an effective, specific, and robust expression cassette that ensures a high level of expression in the mammary glands. In this study, we designed and constructed a series of mammary gland‐specific vectors containing a complex hybrid promoter/enhancer by utilizing promoter sequences from milk proteins (i.e., goat β‐casein, bovine αs1‐casein, or goat β‐lactoglobulin) and cytomegalovirus enhancer sequences; vectors containing a single milk protein promoter served as controls. Chicken β‐globin insulator sequences were also included in some of these vectors. The expression of constructs was analyzed through the generation of transgenic mice. Enzyme‐linked immunosorbent assay (ELISA) analysis revealed that the hybrid promoter/enhancer could drive the expression of recombinant human lactoferrin (rhLF) cDNA at high levels (1.17–8.10 mg/ml) in the milk of transgenic mice, whereas control promoters achieved a very low rhLF expression (7–40 ng/ml). Moreover, the expression of rhLF was not detected in the serum or saliva of any transgenic animal. This result shows that all constructs, driven by the hybrid promoter/enhancer, had high mammary gland‐specific expression pattern. Together, our results suggest that the use of a hybrid promoter/enhancer is a valuable alternative approach for increasing mammary‐specific expression of recombinant hLF in a transgenic mouse model.Mol. Reprod. Dev. 79: 573‐585, 2012. © 2012 Wiley Periodicals, Inc.     

Biosafety evaluation of recombinant protein production in goat mammary gland using adenoviral vectors: preliminary study

The production of recombinant proteins in the milk of non-transgenic goats can be achieved by transducing the mammary gland with recombinant adenoviral vectors. However, this process involves several regulatory issues. The current study evaluates the biosafety of this production system. We present a preliminary biosafety profile based on detection of adenoviral particles in different body fluids and the antibody response after adenoviral transduction of the goat mammary gland. In addition, two methods of adenoviral inactivation in milk were tested. Although adenoviral particles were detected in the milk until day 4 after transduction, they were absent in serum, saliva, urine and feces. Anti-adenovirus antibodies were detected in serum and milk. The virus inactivation methods neutralized adenoviral particles and preserved the immunological identity of the recombinant protein. These results support the idea of a safe production of recombinant proteins using adenoviral vectors.     

Over-expression of human lipoprotein lipase in mouse mammary glands leads to reduction of milk triglyceride and delayed growth of suckling pups

Background

The mammary gland is a conserved site of lipoprotein lipase expression across species and lipoprotein lipase attachment to the luminal surface of mammary gland vascular endothelial cells has been implicated in the direction of circulating triglycerides into milk synthesis during lactation.

Principal Findings

Here we report generation of transgenic mice harboring a human lipoprotein lipase gene driven by a mammary gland-specific promoter. Lipoprotein lipase levels in transgenic milk was raised to 0.16 mg/ml, corresponding to an activity of 8772.95 mU/ml. High lipoprotein lipase activity led to a significant reduction of triglyceride concentration in milk, but other components were largely unchanged. Normal pups fed with transgenic milk showed inferior growth performances compared to those fed with normal milk.

Conclusion

Our study suggests a possibility to reduce the triglyceride content of cow milk using transgenic technology.     

Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors

A key challenge for the academic and biopharmaceutical communities is the rapid and scalable production of recombinant proteins for supporting downstream applications ranging from therapeutic trials to structural genomics efforts. Here, we describe a novel system for the production of recombinant mammalian proteins, including immune receptors, cytokines and antibodies, in a human cell line culture system, often requiring <3 weeks to achieve stable, high-level expression: Daedalus. The inclusion of minimized ubiquitous chromatin opening elements in the transduction vectors is key for preventing genomic silencing and maintaining the stability of decigram levels of expression. This system can bypass the tedious and time-consuming steps of conventional protein production methods by employing the secretion pathway of serum-free adapted human suspension cell lines, such as 293 Freestyle. Using optimized lentiviral vectors, yields of 20-100 mg/l of correctly folded and post-translationally modified, endotoxin-free protein of up to ~70 kDa in size, can be achieved in conventional, small-scale (100 ml) culture. At these yields, most proteins can be purified using a single size-exclusion chromatography step, immediately appropriate for use in structural, biophysical or therapeutic applications.     

The High-Level Expression of Human Tissue Plasminogen Activator in the Milk of Transgenic Mice with Hybrid Gene Locus Strategy

Transgene expression for the mammary gland bioreactor aimed at producing recombinant proteins requires optimized expression vector construction. Previously we presented a hybrid gene locus strategy, which was originally tested with human lactoferrin (hLF) as target transgene, and an extremely high-level expression of rhLF ever been achieved as to 29.8 g/l in mice milk. Here to demonstrate the broad application of this strategy, another 38.4 kb mWAP-htPA hybrid gene locus was constructed, in which the 3-kb genomic coding sequence in the 24-kb mouse whey acidic protein (mWAP) gene locus was substituted by the 17.4-kb genomic coding sequence of human tissue plasminogen activator (htPA), exactly from the start codon to the end codon. Corresponding five transgenic mice lines were generated and the highest expression level of rhtPA in the milk attained as to 3.3 g/l. Our strategy will provide a universal way for the large-scale production of pharmaceutical proteins in the mammary gland of transgenic animals.     

Human parathyroid hormone as a secretory peptide in milk of transgenic mice

In a transgenic mouse model we have targeted the expression of recombinant human parathyroid hormone (hPTH) to the mammary gland yielding hPTH as a secretory, soluble peptide in milk. A 2.5 kb upstream regulatory sequence of the murine whey acidic protein (WAP) directed the expression of the hPTH cDNA in a fusion gene construct (WAPPTHSV2) containing the SV40 small t-antigen intron and polyadenylation site in the 3′ end. Established lines of transgenic mice secreted hPTH to milk in concentrations up to 415 ng/ml. Recombinant hPTH recovered from the milk was purified by HPLC and shown to be identical to hPTH standard as analyzed by SDS-PAGE followed by immunoblotting. Expression of the WAPPTHSV2 was limited to the mammary gland as analyzed by polymerase chain reaction (PCR) and Southern blot of reversed transcribed mRNA from different tissues. hPTH is an important bone anabolic hormone and may be a potentially important pharmaceutical for treatment of demineralization disorders such as osteoporosis. We present the transgenic animal as a possible production system for hPTH. © 1995 Wiley-Liss, Inc.     

Rabbit whey acidic protein gene upstream region controls high-level expression of bovine growth hormone in the mammary gland of transgenic mice

Transgenic mice were produced which secreted high levels of bGH into milk. The 6.3-kb upstream region of the rabbit whey acidic protein (rWAP) gene was linked to the structural part of the bovine growth hormone (bGH) gene, and the chimeric gene was introduced into mouse oocytes. bGH was detected by radioimmunoassay in the milk of all resulting transgenic mice. bGH concentrations in milk varied from line to line, from 1.0–16 mg/ml. This expression was not correlated to the number of transgene copies. In all lines studied, the mammary gland was the major organ expressing bGH mRNA during lactation. bGH mRNA concentrations were barely detectable in the mammary gland of cyclic females; they increased during pregnancy. These results show that the upstream region of the rWAP gene harbors powerful regulatory elements which target high levels of bGH transgene expression to the mammary gland of lactating transgenic mice. © 1995 wiley-Liss, Inc.     

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.     

Lactation performance of transgenic goats expressing recombinant human butyryl-cholinesterase in the milk

The production of recombinant proteins in the milk of transgenic animals has attracted significant interest in the last decade, as a valuable alternative for the production of recombinant proteins that cannot be or are inefficiently produced using conventional systems based on microorganisms or animal cells. Several recombinant proteins of pharmaceutical and biomedical interest have been successfully expressed in high quantities (g/l) in the milk of transgenic animals. However, this productivity may be associated with a compromised mammary physiology resulting, among other things, from the extraordinary demand placed on the mammary secretory cells. In this study we evaluated the lactation performance of a herd of 50 transgenic goats expressing recombinant human butyryl-cholinesterase (rBChE) in the milk. Our findings indicate that high expression levels of rBChE (range 1–5 g/l) are produced in these animals at the expense of an impaired lactation performance. The key features characterizing these transgenic performances were the decreased milk production, the reduced milk fat content which was associated with an apparent disruption in the lipid secretory mechanism at the mammary epithelium level, and a highly increased presence of leukocytes in milk which is not associated with mammary infection. Despite of having a compromised lactation performance, the amount of rBChE produced per transgenic goat represents several orders of magnitude more than the amount of rBChE present in the blood of hundreds of human donors, the only other available source of rBChE for pharmaceutical and biodefense applications. As a result, this development constitutes another successful example in the application of transgenic animal technology.