J. W. Knight

Transgenic pigs as bioreactors: a comparison of gamma-carboxylation of glutamic acid in recombinant human protein C and factor IX by the mammary gland

The mammary gland of transgenic livestock can be used as a bioreactor for producing complex therapeutic proteins. However, the capacity for making a given post-translational modification upon any given polypeptide is uncertain. For example, the efficiency of gamma-carboxylation of glutamic acid in the amino terminal regions of recombinant human protein C (rhPC) and recombinant human Factor IX (rhFIX) is different at similar expression levels. At an expression level of about 200 microg/ml in the milk of transgenic pigs, rhFIX is highly gamma-carboxylated as indicated by pro-coagulant activity and amino acid sequencing. However, only about 20-35% of rhPC has a native, gamma-carboxyglutamic acid-dependent conformation and anti-coagulant activity. Thus, this work provides an example of apparent differences in substrate specificity between two homologous proteins to the endogenous carboxylase of porcine mammary epithelium which leads to varying degrees of post-translational modification.

The Porcine Mammary Gland as a Bioreactor for Complex Proteinsa

The similar biological activity of rhPC and hPC indicates that porcine mammary gland can perform many of the processing reactions necessary for recombinant synthesis of complex human proteins and produce them at levels suitable for industrial bioreactor applications. The health of the transgenic pigs appeared unaffected by the expression of high levels of the heterologous protein. We suggest that one of the advantages of using the mammary gland as a bioreactor appears to be the high cell density relative to that of cell culture.

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 μg ml−1. Within a given outbred lineage having a single loci for the cDNA transgene, the expression levels ranged between 100--400 μg 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

Recombinant human protein C expression in the milk of transgenic pigs and the effect on endogenous milk immunoglobulin and transferrin levels.

Colostrum and milk are natural vehicles for acquiring passive immunity and are valuable tools for decreasing neonatant mortality from diarrheal disease. The effects of recombinant human protein C (rhPC) expression levels on endogenous immunoglobulin and transferrin content of the milk of different lineages of transgenic pigs were studied. The levels of rhPC in the milk ranged from 40 to 1200 μg/ml. Transgenic pigs with rhPC expression levels less than 500 μg/ml had no significant differences in milk protein composition with respect to nontransgenic pigs. A line of transgenic pigs having rhPC expression levels of 960–1200 μg/ml had two- to three-fold higher IgG, IgM, and secretory IgA concentrations compared to other transgenic and nontransgenic pig groups (P < 0.05), and four- to five-fold higher transferrin levels than nontransgenic pigs (P < 0.05). Changes in milk protein composition were not associated with mastitis or other pathologic disruption of epithelial cell junctions as indicated by normal casein and albumin levels in milk. Since IgG, IgM, secretory IgA, and transferrin are transported into the milk by transcytosis, higher levels of these proteins indicate that transcyctosis in the mammary epithelial cell was likely upregulated in pigs having high rhPC expression levels. This study is the first that shows a statistically significant example that mammary tissue specific expression of a heterologous protein can enhance endogenous phenotypic characteristics of milk.

Serum and milk concentrations of leptin in gilts fed a high- or low-energy diet during gestation

Concentrations of leptin in serum and milk were assessed in gilts fed diets during gestation that differed in energy level. Beginning at day 45 and continuing throughout pregnancy, gilts received either a high-energy (6882 kcal metabolizable energy (ME) per day) or low-energy (5221 kcal ME per day) diet (n = 9 per group). All gilts had ad libitum access to a standard lactation diet throughout a 21 day lactation. During gestation, gilts consuming the high-energy diet gained more body weight (P < 0.01) and backfat thickness (P = 0.03) than gilts fed the low-energy diet; however, serum concentrations of leptin remained similar between groups (P = 0.35). Within 24 h after farrowing, gilts fed the high-energy diet had greater levels of leptin in serum and milk than gilts that consumed the low-energy diet during gestation (P < 0.07); Across treatments, backfat thickness and leptin levels in serum were positively correlated (r(2) = 0.51; P = 0.03). At weaning, backfat thickness (P < 0.07), but not body weights or serum and milk levels of leptin (P > 0.1), were greater for gilts fed the high-energy, versus the low-energy, diet during gestation. Gilts that were fed the low-energy diet during gestation consumed more feed during week 2 of lactation (P = 0.06). Our results suggest that altering the level of energy in the diets of gestating swine can influence circulating and milk concentrations of leptin, as well as feed consumption, during lactation.

The effect of lutalyse on the training of sexually inexperienced boars for semen collection

The objective was to determine if i.m. treatments of lutalyse (PGF2alpha; dinoprost tromethamine salt) expedited the training of sexually inexperienced boars for semen collection. Lean-type, terminal-line boars (n = 40; 177.4 +/- 2.4 day of age and 112.8 +/- 2.0 kg body weight) that had not previously experienced natural mating were utilized. Boars were moved individually twice weekly for 6 weeks (total of 12 training sessions) to a semen collection room equipped with an artificial sow. Upon entering the semen collection room, boars received i.m. treatments of either deionized water (4 ml, n = 10) or lutalyse at doses of 5 mg (n = 10), 10 mg (n = 10), or 20 mg (n = 10), and subsequently received a libido score of 1-5 (1 = no interest in the artificial sow; 5 = mounting artificial sow and allowing semen collection). The percentages of boars successfully trained for semen collection during the experimental period were similar (P > 0.05) for controls (20%) and boars receiving 5 mg (30%), 10 mg (20%), or 20 mg (10%) of lutalyse. Average libido score for boars receiving 10 mg lutalyse (2.35 +/- 0.08) was greater (P < 0.05) than for controls (2.14 +/- 0.06). In summary, lutalyse increased libido scores, but did not affect the number of boars trained for semen collection.

Effects of dietary supplementation with an organic source of selenium on characteristics of semen quality and in vitro fertility in boars1

Semen characteristics in boars fed organic or inorganic sources of Se were assessed in 3 experiments. Crossbred boars were randomly assigned at weaning to 1 of 3 dietary treatments: I) basal diets with no supplemental Se (control), II) basal diets with 0.3 mg/kg of supplemental Se from an organic source (Sel-Plex, Alltech Inc., Nicholasville, KY), and III) basal diets supplemented with 0.3 mg/kg of supplemental Se from sodium selenite (Premium Selenium 270, North American Nutrition Co. Inc., Lewisburg, OH). For Exp. 1, semen was collected from boars (n = 10/dietary treatment) on 5 consecutive days at 15 mo of age. Effects of treatment × day were detected for the proportions of progressively motile (P = 0.02) and rapidly moving (P = 0.03) spermatozoa, and measures of sperm velocity, including path velocity of the smoothed cell path (P = 0.05) and average velocity measured in a straight line from the beginning to the end of the track (P = 0.05). Negative effects of day of semen collection on sperm motility were least pronounced in boars fed Sel-Plex. Experiment 2 was conducted when boars were 17 mo of age, and semen was collected (n = 10 boars/dietary treatment), diluted in commercially available extenders, and stored at 18°C for 9 d. Effects of treatment × day were detected for percentages of motile (P = 0.01) and static (P = 0.01) spermatozoa, amplitude of lateral head displacement (P = 0.02), frequency with which the sperm track crossed the sperm path (P = 0.04), straightness (P = 0.01), and average size of all sperm heads (P = 0.03). In general, sperm cells from boars fed Sel-Plex were better able to maintain motility during liquid storage compared with boars fed sodium selenite. For Exp. 3, semen was collected from boars (n = 6/dietary treatment) at 23 mo of age, and spermatozoa were evaluated at d 1 and 8 after semen collection using in vitro fertilization procedures. There was a tendency for an effect (P = 0.11) of dietary treatment on fertilization rate with Sel-Plex-fed boars having the greatest value (70.7%). The results of this study suggest that there are positive effects of dietary supplementation with Sel-Plex on boar semen characteristics and that organic Se supplementation may help ameliorate the negative effects of semen storage on characteristics of sperm motility.

Peripheral plasma testosterone concentration and sexual behavior in young prenatally stressed boars

Abstract The purpose of this study was to examine the effects of stress induced physiological changes in the gestating sow on postnatal sexual and endocrine development of male offspring. Ten boars, ranging from 160 to 185 days of age, were randomly chosen from sows which had been maintained under either stress or control conditions during mid-gestation. Blood samples were collected weekly from each boar (minimum of four weeks) at 30 min intervals over a common six-hour period via an indwelling anterior vena cava cannula. Plasma testosterone concentrations were determined by radioimmunoassay. In order to ascertain degree of sexual behavior, boars were exposed weekly to gilts in estrus and a subjective score assigned. No differences (P>.10) were found between prenatally stressed and control boars in overall mean testosterone concentration or libido score. A significant (P

Rate limitations in posttranslational processing by the mammary gland of transgenic animals

Our studies in transgenic animal bioreactors sought to determine the rate limitations in posttranslational processing of recombinant human protein C (rhPC) made in mammary gland of mice and pigs. Human protein C (hPC) is a complex plasma protein containing nine gamma-carboxylated glutamic acid (gla) residues that bind calcium at about 1 to 3 mM. Gamma carboxylation is a vitamin K-dependent posttranslational modification. The effect of rhPC synthesis rate on the extent of gamma-carboxylation of glutamic acid was studied. We have perturbed the biosynthesis of rhPC by using two different transgenes to direct mammary gland-specific expression. Promoter elements of the murine whey acid protein (mWAP) gene were used to drive the expression of hPC-cDNA and hPC-genomic transgenes. Transgenic mice with hPC-cDNA and hPC-genomic sequences gave expression levels of 11 +/- 4 micrograms rhPC/ml of milk and 895 +/- 21 micrograms rhPC/ml of milk, respectively. Transgenic pigs with hPC-cDNA and hPC-genomic sequences gave expression levels of 100 to 500 micrograms rhPC/ml of milk and 800 to 2000 micrograms rhPC/ml of milk, respectively. A monoclonal antibody (7D7B10-mAb) that binds an epitope in the gla domain of hPC in the absence of calcium was used to study the conformational behavior of immunopurified rhPC. Immunopurified rhPC from lower expressing mice and pigs gave a calcium-dependent binding inhibition by 7D7B10-mAb similar to that of hPC. Immunopurified rhPC from higher expressing mice and pigs gave a less calcium-dependent response. This study suggests that a rate limitation in gamma-carboxylation by the mammary gland occurs at expression levels about > 20 micrograms/ml in mice and > 500 micrograms/ml in pigs.

Relationship between estrone sulfate in plasma and litter size at farrowing for sows and gilts

A positive association (P < 0.01) was detected between estrone sulfate (ES) concentrations in maternal plasma at Day 30 of pregnancy and litter size at parturition in swine. This relationship was best described by a fifth order regression equation (R(2) = 0.5) which indicated that as ES increased from 1 to 7.5 ng/ml on Day 30, litter size increased from 0 (nonpregnant) to 18 piglets farrowed. Day of sampling (P < 0.02), month (P < 0.04) and parity (P < 0.08) were major sources of variation in the model. This indicated that effects of environmental factors such as heat stress, which influence conception rate and embryonic survival, are reflected in changes in maternal ES. Also, larger litter size associated with parous sows is reflected in increased ES in maternal plasma. We conclude that measurement of ES early in gestation may be useful in reproductive management to identify nonpregnant gilts and sows as well as those with small litters.