Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum

Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.     

Changes in the bovine whey proteome during the early lactation period

To investigate time‐dependent change in the bovine whey proteome during the early lactation period, a two‐dimensional gel‐based approach was used in this study. Milk samples were collected from five healthy Friesian‐Holstein dairy cows up to 10 days postpartum. Spot patterns of whey proteins varied drastically from immediately after parturition to 48 h, but no significant changes occurred thereafter. Protein identification by mass spectrometry revealed that the ratios of caseins and immunoglobulins drastically decreased during 48 h postpartum, while those of lower molecular mass proteins such as α‐lactalbumin and β‐lactoglobulin increased. More than 100 spots were detected, being much more abundant in colostral whey than in mature milk whey. Of a total of 25 proteins identified, four, viz. zinc‐α‐2‐glycoprotein, vitamin D‐binding protein, immunoglobulin G2 chain C and β2‐microglobulin, were detectable only in colostrum. Our results indicate that most of the minor whey proteins in colostrum relate to the passive immunity of newborn calves, but some of them play significant roles in nutritional supplementation of the neonate. The characteristics of whey proteins in transition imply that enhancement of innate immunity becomes more important than protection of the neonate against pathogens via passive immunity after 48 h postpartum.     

Aberrant development of mammary glands, but precocious expression of beta-casein in transgenic females ubiquitously expressing whey acidic protein transgene

It has been suggested that whey acidic protein (WAP) may function as a protease inhibitor. However, the actual function of WAP remains obscure. We investigated the histological development of the mammary glands of transgenic mice ubiquitously expressing WAP and CAG/WAP transgene. Ubiquitous expression of WAP induced aberrant development of the lobular alveoli of the mammary glands: mammary alveoli that were either aberrantly large or small in size increased in number in the developing mammary glands of these transgenic females during pregnancy and lactation. The expression of beta-casein was precociously induced in the mammary glands of the transgenic females during early pregnancy and accompanying this was a histological observation that abnormally developed lobular alveoli filled with milk proteins appeared in the mammary glands of transgenic females during early pregnancy. However, during lactation, the development of mammary glands was impaired in transgenic females. To investigate the possible paracrine action of WAP associated with mammary gland aberration, we transplanted the mammary tissue of CAG/EGFP transgenic females into the fat pad of virgin CAG/WAP transgenic females and initiated pregnancy by mating. The development of mammary tissue transplanted to the recipient was histologically examined on day 3 of lactation. The results revealed that the development of grafted mammary tissues was impaired in a manner similar to that of the mammary glands of CAG/WAP transgenic females, indicating that the inhibitory effect of WAP acts via a paracrine mechanism. In vitro experiments using HC11 cells with forced expression of exogenous WAP demonstrated the inhibitory function of WAP on proliferation of mammary epithelial cells.     

Inhibitory function of whey acidic protein in the cell-cycle progression of mouse mammary epithelial cells (EpH4/K6 cells)

Although the biological role for whey acidic protein (WAP) in milk has been suggested, its true function is not known. This paper describes evidence for WAP function in the cell-cycle progression of EpH4/K6 (EpH4), mammary epithelial cells in vitro. The forced expression of exogenous WAP significantly impaired the proliferation of EpH4 cells, whereas it did not affect that of NIH3T3 cells. Apoptosis was not enhanced in the EpH4 cells with stable expression of WAP (WAP-clonal EpH4 cells). The analyses of BrdU incorporation revealed that forced WAP expression significantly reduced incorporation of BrdU in WAP-clonal EpH4 cells compared with control cells transfected with empty plasmid. Among G1 cyclins, the level expression of cyclins D1 was significantly lower in the WAP-clonal EpH4 cells than in control cells. The inhibitory action of WAP on the proliferation of EpH4 cells was enhanced by the presence of extracellular matrix (ECM), but not by the presence of a single component comprising ECM. The cultured medium of WAP-clonal EpH4 cells inhibited the proliferation of control cells without WAP expression. The present results indicate that WAP plays a negative regulatory role in the cell-cycle progression of mammary epithelial cells through an autocrine/paracrine mechanism.     

Analysis of the promoter of mutated human whey acidic protein (WAP) gene

Although whey acidic protein (WAP) has been identified in the milk of a range of species, it has been predicted that WAP is not secreted into human milk as a result of critical point mutations within the coding region. In the present study, we first investigated computationally the promoter region of mutated human WAP genes by comparing with those of other known WAP genes. Computational database analyses showed that the human WAP promoter region was highly conserved, as in other species with milk WAP. Next, we evaluated the activity of the human WAP promoter (2.6 kb) using a reporter gene assay. MCF-7 cells were stably transfected with the hWAP/hGH (human growth hormone) fusion gene, cultured on Matrigel, and treated with lactogenic hormones. Radioimmunoassay detected hGH in the culture medium, indicating that the human WAP promoter was responsible for the lactogenic hormones. The human WAP promoter was significantly more active in MCF-7 cells than the mouse WAP promoter (2.4 kb). The present results provide us with important information on the molecular evolution of milk protein genes.     

Differential patterns of nestin and glial fibrillary acidic protein expression in mouse hippocampus during postnatal development

Intermediate filaments, including nestin and glial fibrillary acidic protein (GFAP), are important for the brain to accommodate neural activities and changes during development. The present study examined the temporal changes of nestin and GFAP protein levels in the postnatal development of the mouse hippocampus. Mouse hippocampi were sampled on postnatal day (PND) 1, 3, 6, 18, and 48. Western blot analysis showed that nestin expression was high at PND 1 and markedly decreased until PND 18. Conversely, GFAP expression was acutely increased in the early phase of postnatal development. Nestin immunoreactivity was localized mainly in the processes of ramified cells at PND 1, but expression subsequently decreased. In contrast, GFAP was evident mainly in the marginal cells of the hippocampus at PND 1, but immunoreactivity revealed satellite, radial, or ramified shapes of the cells from PND 6-48. This study demonstrates that the opposing pattern of nestin and GFAP expressions in mouse hippocampus during postnatal development occur in the early development stage (PND 1-18), suggesting that the opposing change of nestin and GFAP in early postnatal development is important for neural differentiation and positioning in the mouse hippocampus.     

Production of transgenic and non-transgenic clones in miniature pigs by somatic cell nuclear transfer

Miniature pigs have been recognized as valuable experimental animals in various fields such as medical and pharmaceutical research. However, the amount of information on somatic cell cloning in miniature pigs, as well as genetically modified miniature pigs, is much less than that available for common domestic pigs. The objective of the present study was to establish an efficient technique of cloning miniature pigs by somatic cell nuclear transfer. A high pregnancy rate was achieved following transfer of parthenogenetic (3/3) and cloned (5/6) embryos using female miniature pigs in the early pregnancy period as recipients after estrus synchronization with prostaglandin F2 alpha analog and gonadotrophins. The production efficiency of the cloned miniature pigs using male and female fetal fibroblasts as nucleus donors was 0.9% (2/215 and 3/331, respectively). Cloned miniature pigs were also produced efficiently (7.8%, 5/64) by transferring reconstructed embryos into the uteri of common domestic pigs. When donor cells transfected with the green fluorescent protein (GFP) gene were used in nuclear transfer, the production efficiency of the reconstructed embryos and rate of blastocyst development were comparable to those obtained by non-transfected cells. When transfected cell-derived reconstructed embryos were transferred to three common domestic pig recipients, all became pregnant, and a total of ten transgenic cloned miniature pigs were obtained (piglet production efficiency: 2.7%, 10/365). Hence, we were able to establish a practical system for producing cloned and transgenic-cloned miniature pigs with a syngeneic background.     

异种器官移植转基因猪研究进展

1906年,Ｊａｂｏｕｌａｙ将猪肾移植给一位患肾功能衰竭的年青妇女,移植物存活了3ｄ,手术后病人死于尿毒症。此后,外科医生们很快把目标转向同种器官移植(ＡｌｌｏｔｒａｎｓＰｌａｎｔａｔｉｏｎ)。1954年,人类第一例同种肾脏器移植手术获得成功。特别是到了20世纪80年代中期,随着环孢素Ａ的发现,同种器官移植取得了飞速发展。现在,外科医生一般都把移植手术作为心、肝、肾等器官器质性疾病治疗的最终方案。迄今为止,全球已经做了近50万例移植手术,器官移植已成为现代医学领域一个不可缺少的组成部分。在美国,心脏坏死的人数是患爱滋病而死…     

Effect of protein intake during gestation and lactation on the lactational performance of primiparous sows.

The effect of protein intake during gestation and lactation on the lactational performance of primiparous sows was evaluated using 35 Yorkshire x Landrace gilts, allocated to six dietary treatments in a 3 x 2 factorial arrangement. Treatments consisted of three protein levels during gestation, providing approximately 4, 8, and 16 g of lysine/d, and two protein levels (low [L] and high [HI), providing approximately 15 and 45 g of lysine/d, during lactation, respectively. Diets provided similar amounts of ME and all other nutrients. As dietary protein increased during gestation, sows gained more weight and tended to decrease their backfat thickness. There was no gestation x lactation treatment interaction for lactational performance of sows. Feed intake by sows during lactation was usually low but increased (P < .05) with increasing gestation and lactation protein intake and increased linearly (P < .001) as lactation progressed. This linear increase over time was greater (P < .05) in sows fed the H than in sows fed the L protein level. Increased protein intake during lactation reduced (P < .001) 21-d sow weight loss. Milk yield and pig weight gain increased as protein intake increased during gestation (P < .05) and lactation (P < .01). Milk yield did not increase as lactation progressed (P > .05). Pig weight gain increased (P < .05) from wk 1 to 2 of lactation and decreased thereafter. Simple linear regression analysis detected few important relationships between yield of milk components and metabolites or metabolic hormone concentrations. The R2 values for these relationships were < or = .30, except for some relationships between milk component yields and blood urea nitrogen (the range was between .17 and .55). Covariate adjustment for metabolite and metabolic hormone concentrations did not eliminate treatment effects in most cases. This suggests that effects of increased protein intake on milk yield are not fully mediated through metabolite and metabolic hormone concentrations.     

Effects of a whey protein product and spray-dried animal plasma on growth performance of weanling pigs

Five experiments were conducted to evaluate the effects of a high-protein, whey protein product (WPP; 73% CP, 6.8% lysine, 12.8% fat, and 5% lactose) and spray-dried animal plasma (SDAP) on growth performance of weanling pigs. In all experiments, pigs were fed experimental diets from d 0 to 14 after weaning in a pelleted form and then a common diet in meal form for the remainder of the experiment. Dietary treatments were established by substituting WPP or SDAP for dried skim milk (Exp. 1) or soybean meal (Exp. 2, 3, 4, and 5) in the control diet. In Exp. 1, we maintained a constant level of lactose in all diets by adjusting the amount of added crystalline lactose. The amount of lactose in diets used in Exp. 2 through 5 varied slightly by the addition of WPP. In Exp. 1 and 2, 180 weanling pigs (initially 5.8 kg and 19 +/- 1 d of age or 5.5 kg and 17 +/- 1 d of age, respectively) were used. Treatment diets contained SDAP (2.5 and 5%) or WPP (2.7 and 5.4% in Exp.1, and 2.5 or 5.0% in Exp. 2). In Exp. 1, from d 0 to 7 after weaning, ADG and ADFI increased with increasing SDAP (linear, P < .01). No other treatment effects were observed during the d 0 to 14 period. In Exp. 2, from d 0 to 14 after weaning, ADG and G:F increased (linear, P < .04) with increasing SDAP or WWP. In Exp. 3, 305 weanling pigs (initially 4.1 kg and 12 +/- 1 d of age) were used. The control diet contained 2.5% SDAP. The experimental diets were similar to the control diet but contained an additional 2.5 or 5.0% SDAP or 2.5 or 5.0% WPP. From d 0 to 14 after weaning, ADG, ADFI, and G:F increased (quadratic, P < .05) with increasing SDAP up to 5.0%. Increasing WPP increased ADG (quadratic, P < .07) and ADFI (linear, P < .09). In Exp. 4 and 5, 329 and 756 weanling pigs (initially 4.1 kg and 12 +/- 1 d of age and 5.2 kg and 18 +/- 1 d of age, respectively) were fed diets in which WPP was substituted for 0, 25, 50, 75, and 100% (Exp. 4) or 0, 50, and 100% (Exp. 5) of the SDAP in the control diet. In Exp. 4 and 5, from d 0 to 14 after weaning, pigs fed a 1:1 blend of each protein source had better ADG (quadratic, P < .04) than those only fed SDAP. In conclusion, WPP can be used in combination with or as a total replacement for SDAP in diets for weanling pigs without reducing performance.     

Energy concentration and phosphorus digestibility in whey powder, whey permeate, and low-ash whey permeate fed to weanling pigs

Two experiments were conducted to determine DE and ME, the apparent total tract digestibility (ATTD) of P, and the standardized total tract digestibility (STTD) of P in whey powder (3,646 kcal/kg), whey permeate (3,426 kcal/kg), and low-ash whey permeate (3,657 kcal/kg) fed to weanling pigs. The DE and ME in the 3 whey products were determined using 32 barrows (9.2 ± 0.4 kg of BW). A basal diet based on corn, soybean meal, and fish meal and 3 diets containing 70% of the basal diet and 30% of each whey product were prepared. Each diet was fed to 8 pigs that were housed individually in metabolism cages. The total collection method was used for fecal and urine collections with 5-d adaptation and 5-d collection periods, and the difference procedure was used to calculate DE and ME in the 3 whey products. The concentrations of DE in whey powder and low-ash whey permeate were greater (P < 0.001) than in whey permeate (3,646 and 3,683 vs. 3,253 kcal/kg of DM). The concentrations of ME in whey powder and low-ash whey permeate were also greater (P < 0.001) than in whey permeate (3,462 and 3,593 vs. 3,081 kcal/kg of DM). The ATTD and STTD of P in the 3 whey products were determined using 32 barrows (11.0 ± 0.81 kg of BW). Three cornstarch-sucrose-based diets containing 30% of each whey product as the sole source of P were prepared. A P-free diet that was used to estimate the basal endogenous losses of P was also formulated. The ATTD of P in whey powder and in whey permeate was greater (P < 0.001) than in low-ash whey permeate (84.3 and 86.1 vs. 55.9%), but the STTD values for P were not different among the 3 ingredients (91.2, 93.1, and 91.8% in whey powder, whey permeate, and low-ash whey permeate, respectively). In conclusion, whey permeate contains less GE, DE, and ME than whey powder and low-ash whey permeate, but all 3 ingredients have an excellent digestibility of P.     

Parturition body size and body protein loss during lactation influence performance during lactation and ovarian function at weaning in first-parity sows

We investigated the effect of body protein mass at parturition and different degrees of body protein loss in lactation on sow performance. In a 2 x 2 factorial arrangement, 77 Genex gilts were fed to achieve either a standard or high body mass at parturition and to lose either a moderate (MPL) or high (HPL) amount of protein in lactation. Pregnant gilts were fed either 24.4 MJ of ME, 266 g of CP, and 11 g of lysine/d or 34.0 MJ of ME, 436 g of CP, and 20 g of lysine/d resulting in divergent (P < 0.01) live weights (165 vs. 193 kg) and calculated protein masses (24.3 vs. 30.0 kg) and slightly different backfat depths (20.0 vs. 22.8 mm; P < 0.05) at parturition. Diets fed during lactation were formulated to deliver 731 g of CP and 37 g of lysine/d or 416 g of CP and 22 g of lysine/d to induce differential body protein mobilization. Sows were slaughtered at weaning (d 26), and the weight of the organs and the lean, fat, and bone in five primal cuts was measured. The external diameter of the eight largest follicles on each ovary was recorded, and the follicular fluid from these follicles was collected, weighed, and analyzed for estradiol. Losses in lactational live weight (26 vs. 20 kg; P < 0.01) and calculated protein mass (17.8 vs. 10.7%; P < 0.001) were greater, and the carcass lean mass at weaning was 10% lighter (P < 0.05) in HPL sows. Backfat (5.1 +/- 0.8 mm; P = 0.29) and calculated fat mass (25.8 +/- 1.5%; P = 0.84) losses did not differ between treatments. Both sow body mass (P < 0.05) and lactation protein loss (P < 0.01) affected litter growth rate. Litter growth rate decreased (P < 0.05) at the end of lactation in HPL sows once these sows had lost 10 to 12% of their calculated protein mass. Ovarian follicular development was most advanced in high body mass sows that lost the least protein; these sows had the heaviest (P < 0.05) uterine weight and highest (P < 0.05) follicular fluid estradiol concentration. Follicular development was least advanced in standard body mass sows that lost the most protein. These sows had the lowest (P < 0.05) muscle:bone ratio at weaning and likely lost the largest proportion of their muscle mass compared with the other treatments. In conclusion, ovarian function at weaning and litter performance was higher in high body mass sows and in sows that lost the least protein in lactation, suggesting that a larger lean mass may delay the onset of a decrease in performance in sows that lose protein in lactation.     

Synthesis and secretion of A-type natriuretic peptide in the auricular cardiocytes during pregnancy and lactation in mouse

A-type (atrial) natriuretic peptide (ANP) levels in the auricular cardiocytes and plasma were examined by immunohistochemistry, electron microscopy, and radioimmunoassay in pregnant and lactating mice. Additionally, the cardiocyte ANP mRNA expression was measured by the polymerase chain reaction method. ANP-immunoreactivity (IR) and the number of ANP-granules in the cardiocytes on the 18th day of gestation were greater than those in virgin controls, but the plasma ANP concentration decreased on the 18th day of gestation. On the day of delivery, ANP-IR and the number of ANP-granules in the cardiocytes were decreased compared to those during the pregnancy and to those in virgin controls, and then began to increase continually until the 15th day of lactation. Plasma ANP concentration after delivery was significantly higher than that during pregnancy, and than that in virgin controls, and continued to increase until the 15th day of lactation. Cardiocyte ANP mRNA expression was highest on the day of delivery compared to that in all the other times. In conclusion, these results suggested that the circulating systems of ANP during pregnancy and lactation were regulated differentially.