Transgenic Goats in the World Pharmaceutical Industry of the 21st Century

In many developed countries, isolation of human pharmaceutical proteins from milk of genetically modified animals is currently a priority. One of the first commercial pharmaceuticals obtained from the milk of transgenic goats, an anticoagulant antithrombin III, developed by Genzyme Transgenic Corporation, an American biotechnological company, will appear on the pharmaceutical market in the nearest future. In this review, we discuss the role of fundamental science in the development of this field of the pharmaceutical industry.     

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.     

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.     

Alcohol Stability of Milk from the Perspective of X-Ray Diffractometry

The stability of alcohol solutions is an issue that is still important for the dairy industry. The crystallographic profile of milk that is stable or unstable to alcohol has never previously been evaluated by X-ray diffractometry. In order to provide more information about aspects of crystallinity, we investigated the crystallographic profile of milk that was stable or unstable in 72 % (v/v) alcohol solution. The samples were lyophilized and analyzed by diffractometer and scanning electron microscopy. The unstable milk samples showed higher relative crystallinity and more intense peaks in the X-ray spectra. This suggested that the crystallographic structure of the milk that was unstable to alcohol may have been different to the stable milk. The intensity of the peaks could be explained by the quantity of α-lactose crystals, which were present in a greater ratio in the unstable milk, while the stable milk presented other lactose-crystal forms, such as β-lactose or crystals of α and b-lactose in different molar ratios. The findings of this study reinforce the fact that the alcohol stability test is not always a reliable indicator in predicting the microbiological origin of acidity and thermal stability. In terms of food and pharmaceutical technology, the results of this study provide another variable that is useful to control the alcohol stability of any product containing ethanol and lactose, i.e. the ratio between the polymorphisms of lactose.     

Methods for the quantitation of human milk oligosaccharides in bacterial fermentation by mass spectrometry

Oligosaccharides are the third most abundant component in human milk. In the past decades, it became apparent that they would be able to protect against pathogens and participate in the development of the gut microflora for infants. However, their role in infants' nutrition and development remains poorly understood. To better understand this function, it is extremely important to have a quantitative tool for profiling oligosaccharides. In this article, we show the development of a method to quantitatively differentiate the relative amounts of oligosaccharides fermented by different intestinal bacteria. To determine the oligosaccharide consumption, bacteria were grown in a medium using human milk oligosaccharides (HMOs) as the only carbon source purified from breast milk and further analyzed by matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS). A method using an internal deuterium-labeled standard was developed and compared with an external standard method, with the internal standard method giving better precision and unambiguous measurements than the external standard method and providing to be a novel and robust tool for following bacterial fermentation of milk oligosaccharides.     

Lactose intolerance in captive nocturnal prosimians (Perodicticus potto): A twenty-one year record

A colony of pottos was captured in December 1959 and brought to the United States. At that time part of their diet consisted of a high protein porridge made with whole milk. In addition, their drinking water contained an antibiotic to protect them from possible infection resulting from the change in habitat. No intestinal incontinence resulted from this treatment. Antibiotic addition to the diet ceased in 1961. In 1971 the remaining pair of animals developed a calcium deficiency. This was alleviated by adding 5 g calcium lactate to the drinking water and serving whole milk every other day. In 1977 the male developed acute lactose intolerance. His feces became bacteriologically sterile. His mate, who died on January 9, 1979, produced feces containing enteric organisms throughout her life. A large number of dairy products were served the animals in the attempt to alleviate this problem. Acidophilus milk or tinned 2% milkfat containing milk with 100% of the lactose hydrolyzed, produced no intestinal incontinence in the animal. It is suggested that sterile stools resulted from a fungal infection that killed theEscherichia coli. In addition it is proposed that non-pathogenic bacteria brought about an alteration of the brush border of the columnar epithelial cells of the villi which synthesize the enzyme lactase, such that lactase production was seriously reduced.     

Transgenesis for the study and the control of lactation

The study and the control of milk synthesis are required to decipher the mechanisms of gene expression, to improve milk production, to modify milk composition, to induce a resistance to diseases in the mammary gland and to produce recombinant proteins of pharmaceutical interest. Transgenesis has become a mandatory tool to reach these goals. The use of transgenesis is still limited by the difficulty of adding foreign genes in farm animals and mainly by replacing genes by homologous recombination. Transgene expression is also often ill-controlled. The present paper summarizes the current progress in this field with a particular emphasis on expression vectors for transgenes.     

Prediction of first test day milk yield using historical records in dairy cows

The transition between two lactations remains one of the most critical periods during the productive life of dairy cows. In this study, we aimed to develop a model that predicts the milk yield of dairy cows from test day milk yield data collected in the previous lactation. In the past, data routinely collected in the context of herd improvement programmes on dairy farms have been used to provide insights in the health status of animals or for genetic evaluations. Typically, only data from the current lactation is used, comparing expected (i.e., unperturbed) with realised milk yields. This approach cannot be used to monitor the transition period due to the lack of unperturbed milk yields at the start of a lactation. For multiparous cows, an opportunity lies in the use of data from the previous lactation to predict the expected production of the next one. We developed a methodology to predict the first test day milk yield after calving using information from the previous lactation. To this end, three random forest models (nextMILK_FULL, nextMILK_PH, and nextMILK_P) were trained with three different feature sets to forecast the milk yield on the first test day of the next lactation. To evaluate the added value of using a machine-learning approach against simple models based on contemporary animals or production in the previous lactation, we compared the nextMILK models with four benchmark models. The nextMILK models had an RMSE ranging from 6.08 to 6.24 kg of milk. In conclusion, the nextMILK models had a better prediction performance compared to the benchmark models. Application-wise, the proposed methodology could be part of a monitoring tool tailored towards the transition period. Future research should focus on validation of the developed methodology within such tool.     

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.     

A novel approach to the recovery of biologically active oligosaccharides from milk using a combination of enzymatic treatment and nanofiltration

A new easily scalable approach to the recovery of biologically active oligosaccharides from milk has been developed which relies on the combination of enzymatic treatment of defatted milk using beta-galactosidase and nanofiltration. It was shown that enzymatic hydrolysis of lactose significantly improves the efficiency and selectivity of membrane-based separations. With the best membrane, as much as 6.7 g of oligosaccharides (containing very little contaminating lactose) could be obtained from one liter of defatted human milk in just four nanofiltration cycles. The human milk oligosaccharides recovered by this method were shown to inhibit binding of intimin, an adhesion molecule of enteropathogenic Escherichia coli, to epithelial cells in vitro. No significant difference in the oligosaccharide profile between samples prepared by this method and conventional gel-permeation chromatography was found. The developed approach is also suitable for the recovery of substantial quantities of tri- and tetra-saccharides from caprine milk.     

Preparation of recombinant proteins in milk to improve human and animal health

Milk is a very abundant source of proteins for animal and human consumption. Milk composition can be modified using transgenesis, including exogenous gene addition and endogenous gene inactivation. The study of milk protein genes has provided researchers with regulatory regions capable of efficiently and specifically driving the expression of foreign genes in milk. The projects underway are aimed at modifying milk composition, improving its nutritional value, reducing mammary infections, providing consumers with antipathogen proteins and preparing purified recombinant proteins for pharmaceutical use. The present paper summarises the current progress in this field.     

A modification of lactoferrin isolation method]

A modified method was developed to isolate lactoferrin from women's colostrum by means of ion-exchange chromatography on a Servacel CM-52 column and gel filtration on a Toyoperal HW-52 column. These changes simplified the basic method and decreased the length of the procedure without affecting the biological activity and purity of the preparation. The modified method can be recommended to produce lactoferrin suitable for the use as a pharmaceutical preparation and nutritional additive to milk substitutes.     

Protein profile and alpha-lactalbumin concentration in the milk of standard and transgenic goats expressing recombinant human butyrylcholinesterase

The expression of recombinant proteins of pharmaceutical interest in the milk of transgenic farm animals can result in phenotypes exhibiting compromised lactation performance, as a result of the extraordinary demand placed on the mammary gland. In this study, we investigated differences in the protein composition of milk from control and transgenic goats expressing recombinant human butyrylcholinesterase. In Experiment 1, the milk was characterized by gel electrophoresis and liquid chromatography/mass spectrometry in order to identify protein bands that were uniquely visible in the transgenic milk and/or at differing band densities compared with controls. Differences in protein content were additionally evaluated by computer assisted band densitometry. Proteins identified in the transgenic milk only included serum proteins (i.e. complement component 3b, ceruloplasmin), a cytoskeleton protein (i.e. actin) and a stress-induced protein (94 kDA glucose-regulated protein). Proteins exhibiting evident differences in band density between the transgenic and control groups included immunoglobulins, serum albumin, β-lactoglobulin and α-lactalbumin. These results were found to be indicative of compromised epithelial tight junctions, premature mammary cell death, and protein synthesis stress resulting from transgene expression. In Experiment 2, the concentration of α-lactalbumin was determined using the IDRing^® assay and was found to be significantly reduced on day 1 of lactation in transgenic goats (4.33 ± 0.97 vs. 2.24 ± 0.25 mg/ml, P < 0.01), but was not different from non-transgenic controls by day 30 (0.99 ± 0.46 vs. 0.90 ± 0.11 mg/ml, P > 0.05). We concluded that a decreased/delayed expression of the α-lactalbumin gene may be the cause for the delayed start of milk production observed in this herd of transgenic goats.     

Expression of recombinant Hirudin in transgenic mice milk driven by the goat beta-casein promoter

Hirudin, isolated from the leech Hirudo medicinalis, inhibits thrombin directly and several expression systems have been used to produce recombinant Hirudin (rHirudin) for pharmaceutical purposes. A DNA fragment containing the Hirudin coding sequence and goat beta-casein secretion signal was chemically synthesized in this study. The synthetic DNA then was further constructed into a goat beta-casein expression vector for mouse transgenesis. Four lines of transgenic mice were successfully developed and one line showed a meaningful anti-thrombin activity of 40,000 anti-thrombin units (ATU)/mL in their milk. In this animal line, Hirudin mRNA was found in samples of uterus and kidney with insignificant anti-thrombin activity (相似文献   

A PCR method for detection of bifidobacteria in raw milk and raw milk cheese: comparison with culture-based methods

Bifidobacteria are well known for their beneficial effects on health and are used as probiotics in food and pharmaceutical products. As they form one of the most important groups in both human and animal feces, their use as fecal indicator organisms in raw milk products has recently been proposed. Bifidobacteria species isolated in humans are different from those isolated in animals. It should therefore be possible to determine contamination origin (human or animal). A method of detecting the Bifidobacterium genus was developed by PCR targeting the hsp60 gene. The genus Bifidobacterium was identified by PCR amplification of a 217-bp hsp60 gene fragment. The degenerated primer pair specific to the Bifidobacterium genus used was tested for it specificity on 127 strains. Sensitivity was measured on artificially contaminated samples. Food can however be a difficult matrix for PCR testing since it contains PCR inhibitors. So an internal PCR control was used. An artificially created DNA fragment of 315 bp was constructed. The PCR detection method was tested on raw milk and cheese samples and compared with three culture-based methods, which comprised enrichment and isolation steps. The enrichment step used Brain Heart Infusion medium with propionic acid, iron citrate, yeast extract, supplemented with mupirocin (BHMup) or not (BH) and the isolation step used Columbia blood agar medium, supplemented with mupirocin (CMup) or not (C). The method using mupirocin at both enrichment and isolation steps and the PCR method performed from the culture in BHMup enrichment medium were shown to be the most efficient. No significant difference was observed in raw milk samples between PCR from BHMup and the culture-based method BHMup/CMup, while a significant difference was noticed between the same methods in raw milk cheese samples, which would favor using PCR. The results suggested that PCR on the hsp60 gene was convenient for a rapid detection of bifidobacteria in raw milk and raw milk cheese samples and that bifidobacteria always present throughout raw milk cheese production could be efficiently used as fecal indicators.     

A solid-phase radioimmunoassay for progesterone and its application to pregnancy diagnosis in the cow

A solid-phase radioimmunoassay using antibody-coated tubes and ¹²⁵I-progesterone label was developed to provide an alternative to the conventional aqueous method of assaying progesterone in milk for early pregnancy diagnosis. The accuracy of diagnoses made using the solid-phase assay of progesterone in milk was assessed by comparison with diagnoses made using an aqueous assay of serum progesterone. Both methods agreed in the thirteen cows that were diagnosed. When “fat-free” milk was assayed by both aqueous (x) and solid-phase (y) methods, the progesterone values which resulted showed a high correlation (r = .94) and a linear relationship of y = 1.67x ? 0.68. Milk samples, in which the fat concentration ranged from 0.20 to 4.04%, were assayed by the solid-phase method and no relationship between milk fat and progesterone concentration was observed. Pregnancy diagnosis from solid-phase assay of milk samples collected on the day of breeding, 21 and 23 days following breeding was performed on 62 Holstein cattle. The accuracy of “non-pregnant” diagnoses was 95% to 100% and the accuracy of “pregnant” diagnoses was 80% if breeding day values were used and 72% if these values were excluded.The accuracy of this assay in diagnosing pregnancy was equal to that of previously published assays and provided the advantages of requiring less technical time and equipment. In addition, foremilk, composite or stripping samples can be accomodated in this assay since the estimates of progesterone are not affected by the concentration of milk fat.     

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.     

Jennet milk production during the lactation in a Sicilian farming system

In Italy, the interest for jennet milk production has recently developed. An 18-month-long experiment was carried out on a jennet farm near Milo (CT), where 24 jennets, which derived from the Ragusana breed, were tested for milk yield and composition over an entire lactation period. The jennets were fed with hay and concentrate in a large paddock. From the 28th post-foaling day to the end of the lactation, the jennets were machine-milked twice a day with an in-between milking interval of 5 h. The milk amount from each jennet was recorded every 3 weeks and individual samples were collected and analyzed for fat, protein, casein, non-proteic nitrogen, lactose and somatic cell count. This study showed that jennets at Sicilian latitudes are not seasonal polyestrous. The daily milk yield, the length of lactation and the milk characteristics varied depending on the foaling season. The total average milk production was 490 ± 36 kg in 295 ± 12 post-foaling days, considering two milking records per day. During the lactation, milk yield decreased constantly from 1.98 to 1.28 kg/jennet per day. When looking at the jennet milk quality during lactation, the percentage of fat and protein decreased, while the lactose percentage increased, according to a tendency apparently unique for equines when compared to the ruminants. When looking at the productive season, spring generally gave the best qualitative and quantitative results. Based on these results, jennet milk yield and quality could be improved; furthermore, jennet milk production may turn out to be a profitable business.