Timing of DNA integration, transgenic mosaicism, and pronuclear microinjection.

Selection of transgenic embryos prior to embryo transfer is a means to increase the efficiency of transgenic livestock production. Among transgenic reporters, cytoplasmic expression of green fluorescent protein (GFP) has features that make it ideal for transgenic embryo selection. The primary objective of this study was to assess cytoplasmic expression of a specially designed GFP reporter as a tool for transgenic bovine embryo selection. A second objective was to evaluate this reporter for studying transgenic mosaicism related to timing of integration of pronuclear microinjected DNA. Transgenic embryos produced by pronuclear injection showed a discrete pattern of GFP expression with clusters at 25, 50, and 100% of blastomeres expressing GFP. This pattern of mosaicism is interpreted to indicate that the integration of microinjected DNA occurred, not only at the pronuclear stage, but also in the subsequent cell divisions. Among the GFP-positive transgenic embryos, only in 21% did all the blastomeres show the green fluorescence. Using the fraction of positive blastomeres within an embryo, the timing of integration of microinjected DNA was estimated. The frequency of nonmosaic embryos expressing GFP is consistent with published germline transmission success rates of transgenic cattle derived from pronuclear microinjected embryos. These results indicate the possible application of GFP as a marker of transgenic embryos and graphically illustrate underlying complexities in DNA integration in embryos subjected to pronuclear microinjection.

Transgenic cattle produced by reverse-transcribed gene transfer in oocytes.

A critical requirement for integration of retroviruses, other than HIV and possibly related lentiviruses, is the breakdown of the nuclear envelope during mitosis. Nuclear envelope breakdown occurs during mitotic M-phase, the envelope reforming immediately after cell division, thereby permitting the translocation of the retroviral preintegration complex into the nucleus and enabling integration to proceed. In the oocyte, during metaphase II (MII) of the second meiosis, the nuclear envelope is also absent and the oocyte remains in MII arrest for a much longer period of time compared with M-phase in a somatic cell. Pseudotyped replication-defective retroviral vector was injected into the perivitelline space of bovine oocytes during MII. We show that reverse-transcribed gene transfer can take place in an oocyte in MII arrest of meiosis, leading to production of offspring, the majority of which are transgenic. We discuss the implications of this mechanism both as a means of production of transgenic livestock and as a model for naturally occurring recursive transgenesis.

Factors affecting herd milk composition and milk plasmin at four levels of somatic cell counts.

A longitudinal study was carried out on samples of bulk tank milk collected from 200 farms for 12 mo to evaluate SCC, plasmin and plasminogen activities, psychrotrophic bacteria count, and protein quality in milk and to examine the proteolytic effects of plasmin on milk proteins. Herds were selected randomly from client lists of two dairies to create four groups based on milk SCC of the month before the study; herds were reassigned monthly to one of four groups based on SCC for that month. Overall means were 3.73% fat, 3.13% protein by infrared analysis, 3.16% protein by Kjeldahl analysis, 2.42% casein percentage, 4.65% lactose, 5.43 cells/ml of log SCC, 1.13 U of plasmin, 45.6 U of plasminogen, and log 2.86 cfu/ml of psychrotrophic bacteria. The ANOVA showed a significant effect of month on all factors except SCC, which was fixed by the experimental design. Plasmin and plasminogen activities were high from December to May. Plasmin activities and psychrotroph counts were significantly higher for the high SCC group. Casein percentage and number were significantly higher for the low SCC group.

Prediction of milk yields from serum beta-lactoglobulin concentrations in pregnant heifers.

The serum concentration of beta-LG at wk 26 of pregnancy was determined in Holstein heifers of known genetic merit to evaluate use of a single blood sample as an index to predict their subsequent milk yield. Concentrations of beta-LG varied widely (16 to 1200 ng/ml) in the serum of the heifers at this stage of pregnancy. Subsequent measures of lactation performance showed that heifers with high beta-LG concentrations in pregnancy produced more milk than those with low beta-LG in serum. Previous studies have shown that the concentration of beta-LG in serum shows a log-linear increase during pregnancy, which is consistent with correlation to the growth of the mammary parenchyma. Therefore, the logarithm beta-LG concentration was used as the index in this study. The correlations of this index were .46, .54, and .48 for first lactation 305-d yields of milk, fat, and protein, respectively. The results suggest that serum concentrations of beta-LG estimated from a single blood sample at this stage of pregnancy can be used to predict milk yield. Analysis showed that an explanation of future milk yield was significantly improved by use of serum beta-LG and parent average. Serum beta-LG can be used as a physiological marker to prescreen heifers for lactation potential.

Variation in expression of a bovine alpha-lactalbumin transgene in milk of transgenic mice.

Transgenic mice were produced to study the production of bovine alpha-LA in their milk. A 7.6-kb fragment containing a bovine alpha-LA gene was purified and microinjected into pronuclear stage mouse embryos. This fragment contained 2.0 kb of 5' flanking region, the 1.7-kb coding region, and 2.7 kb of 3' flanking region. Out of 121 potential transgenic founder mice, 3 were identified as being transgenic by the polymerase chain reaction. Multiple mice from the second, third, and fourth generation from each line were milked, and the milk was analyzed using an ELISA assay and Western blots to determine the presence of bovine alpha-LA. Bovine alpha-LA was present at concentrations up to 1.5 mg of protein/ml of mouse milk. The high degree of expression variation between mice within each of the transgenic lines was a characteristic that has not been reported in other studies of transgene expression in milk. Production of bovine alpha-LA in the milk of these transgenic mice showed a high degree of variation both within a lactation and between mice within a line. The bovine alpha-LA concentration in a single line of transgenic mice exhibited as much as a 10-fold variation between mice. Variations as high as 3-fold were detected within a single lactation in the same mouse. These differences in expression appeared to be correlated with mouse milk production; bovine alpha-LA was higher on d 10 and 15 of lactation than on d 5. Transgenic mice that show variation in expression of a bovine gene might offer a unique system for studying quantitative traits in a laboratory model.

Human growth hormone (hGH) secretion in milk of goats after direct transfer of the hGH gene into the mammary gland by using replication-defective retrovirus vectors.

Mammary-specific promoters have been used in transgenic animals to limit transgene expression to the mammary gland. Gene therapy techniques to target just one organ for introduction of a foreign gene have also been demonstrated. We have directly infused replication-defective retroviruses encoding hGH into the mammary gland of goats via the teat canal during a period of hormone-induced mammogenesis. This resulted in the secretion of hGH into the milk when lactation commenced on day 14 of the regime. Levels of hGH in the milk were highest on the first day of lactation, averaging approximately 60 ng/ml, and declined to a plateau of 12 ng/ml from day 9 to day 15 of lactation. Thus we report targeting of replication-defective retroviruses to the mammary secretory epithelial cells to produce foreign proteins in the milk of ruminants.

Genetic engineering of milk composition: modification of milk components in lactating transgenic animals.

Recent progress in recombinant DNA technology as well as in embryo manipulation and transfer has made the introduction of specific genes into the germline of animals relatively commonplace. With appropriate genetic constructs expression of the inserted genes in transgenic animals can be controlled in a tissue-specific and in a differentiation-specific manner; thus, it is now possible to consider alteration of the composition of milk produced by a lactating animal in any of a variety of ways. There is a growing list of foreign milk proteins that have been expressed, and one can envisage placing almost any protein gene of interest under the control of the cis-acting promoter and enhancer elements of a milk protein gene. Modification of milk composition can be extended not only to the proteins of commodity value but also, by manipulation of key metabolic enzymes, to fat, lactose, and other minerals in milk.

Correlation of the alpha-lactalbumin (+15) polymorphism to milk production and milk composition of Holsteins.

The alpha-lactalbumin (+15) polymorphism (a single base variation 15 basepairs 3' of the alpha-lactalbumin transcription start point) was examined for its usefulness as a genetic marker for Holsteins. The +15 polymorphism is located in a region of the gene that is potentially involved in the regulation of alpha-lactalbumin gene expression. Animals from two dairy herds and young sires from progeny-testing programs of four AI organizations were used in the analysis. A group of sons from a heterozygous sire were also evaluated. Each individual animal was genotyped at the alpha-lactalbumin (+15) locus, and differences of genotypes were investigated. Estimated differences among alleles were calculated for PTA for milk, kilograms of protein, protein percentage, protein dollars, kilograms of fat, fat percentage, and fat dollars. Animals having the alpha-lactalbumin (+15) AA (an adenine on both alleles at position +15) genotype had statistically higher PTA for milk, kilograms of protein, protein dollars, kilograms of fat, and fat dollars than did the alpha-lactalbumin (+15) BB (a cytosine, guanine, or thymine on both alleles at position +15) animals. The alpha-lactalbumin (+15) BB animals had higher protein and fat percentages than the alpha-lactalbumin (+15) AA animals. Animals that were heterozygous at this locus, alpha-lactalbumin (+15) AB, had intermediate values for all traits analyzed. These results indicate a potential marker or actual locus effect of the alpha-lactalbumin (+15) polymorphism in Holstein cattle.

The effects of daily oxytocin injections before and after milking on milk production, milk plasmin, and milk composition.

Exogenous daily oxytocin injections given immediately before milking increase milk production. To investigate the mechanism by which oxytocin increases milk production, oxytocin injections were given before and after milking, and saline injection was given before milking as a control. The experimental design was a replicated Latin square; two complete trials were performed: one with 12 cows (45 d) and another with 15 cows (95 d). In the first trial, the least squares means of milk production were 29.2, 29.3, and 28.3 kg for oxytocin injection before milking, oxytocin injection after milking, and saline injection before milking, respectively. In the second trial, the least squares means of milk production were 33.3, 32.9, and 32.4 kg for oxytocin injection before milking, oxytocin injection after milking, and saline injection before milking, respectively. Oxytocin before and after milking significantly increased milk production by 3%. The results suggest that increases in milk production may not be caused by removal of residual milk but by increased gland output of milk. The effect on milk plasmin activity, fat, protein, SCC, and lactose was nonsignificant and may indicate that effect of oxytocin is not manifested through an effect on cell remodeling.

Sequence and single-base polymorphisms of the bovine alpha-lactalbumin 5'-flanking region.

The alpha-lactalbumin (alpha LA)-encoding gene is a potential quantitative trait locus in dairy animals. In cattle, the production of alpha LA is tightly coupled to the onset of lactation and it serves as a regulatory subunit of the enzyme responsible for lactose synthesis. Lactose is the major osmole controlling water movement in the mammary gland. To better understand the control of bovine alpha LA expression, the 5'-flanking region of a Holstein alpha LA gene was cloned and sequenced. The sequenced clone contains 1952 bp of 5'-flanking region and 66-bp of the protein-coding region. Three single-bp polymorphisms were identified within this region. These polymorphisms occur at positions +15, +21 and +54 relative to the mRNA transcription start point (tsp). The +15 and +21 variations occur in the region encoding the 5'-untranslated region of the mRNA-coding sequence. The +54 polymorphism is a silent mutation in the SP-coding region of the gene. A polymerase chain reaction (PCR, Cetus)-based screening method has been employed to analyze the genotype of cattle at the +15 position. A total of 501 randomly selected cattle from seven breeds were screened for this allele. Of these animals, only the Holstein breed of cattle was found to contain the +15 variation and it occurs at a gene frequency of 32%. Sequence comparisons were conducted between the 5'-flanking regions of the bovine-milk-protein encoding genes, alpha LA, beta-casein and alpha S1-casein, which are coordinately expressed. Regions of similarity extending to 350 bp in length were observed between these sequences.

Xerographic paper as a transfer medium for western blots: quantification of bovine alpha S1-casein by western blot.

We have found that xerographic paper (regular photocopy or laser printer paper) can be used as a transfer medium for protein blots for the immunodetection of low concentrations (a few nanograms) of bovine alpha S1-casein. With paper blotting, we could detect the protein with three times more sensitivity than with polyvinylidene difluoride. Blotting was achieved by transfer from sodium dodecyl sulfate-polyacrylamide gel electrophoresis to the methanol-wet paper. The blot was incubated with chicken anti-casein antibodies, sequentially peroxidase-labeled rabbit anti-chicken antibodies, and diaminobenzidine substrate. The blots were directly scanned and the pixel intensity of the band areas was integrated. An analysis of the scanned blots showed that the log of protein concentration was linearly related with the square root of an integrated pixel intensity (r2 greater than 0.96). This linear relationship was observed in a wide range of protein concentration from 5 ng to 15 micrograms. The coefficients of variation were 12.4% for intraassay and 15.7% for interassay. This new analytical procedure can be applied to estimate the concentration of proteins by immunological blotting.

Enzyme-linked immunosorbent assays for bovine alpha-lactalbumin and beta-lactoglobulin in serum and tissue culture media.

Enzyme-linked immunosorbent assays for bovine alpha-lactalbumin and beta-lactoglobulin have been developed for measurements of serum and tissue culture samples. Either alpha-lactalbumin or beta-lactoglobulin antiserum was coated on ELISA plates. Biotinylated proteins were used in competition with unknown amount of proteins in samples. After unbound proteins were washed off, ExtrAvidin-peroxidase and tetramethylbenzidine were then used as a detection system. Crossreactivity of caseins or bovine serum albumin was less than .0001% in either alpha-lactalbumin or beta-lactoglobulin ELISA. Parallel curves from serial dilutions were obtained in serum and media samples. The additivity of alpha-lactalbumin and beta-lactoglobulin ELISA was validated in either serum or medium samples. The intraassay and interassay coefficients of variation for alpha-lactalbumin and beta-lactoglobulin ELISA were below 10% over 51 and 47 assays. The ELISA are useful in mammary gland biology studies for measuring milk whey protein in serum or culture media.

Serum concentrations of the milk proteins alpha-lactalbumin and beta-lactoglobulin in pregnancy and lactation: correlations with milk and fat yields in dairy cattle.

Serum concentrations of alpha-lactalbumin and beta-lactoglobulin in first pregnancy, parturition, lactation, involution, and second parturition in 37 Holstein cattle were determined and used as an index of mammary status and in predicting milk yield. During first pregnancy, serum alpha-lactalbumin increased in the last 3 mo and reached a peak at parturition (approximately 1100 ng/ml). Changes in alpha-lactalbumin could not be described by a simple exponential equation, whereas changes in serum beta-lactoglobulin were described by a single exponential from second trimester until 4 wk prepartum and reached a peak at parturition (approximately 460 ng/ml). By 2 wk after parturition, alpha-lactalbumin had dropped to approximately 140 ng/ml, and beta-lactoglobulin dropped to approximately 25 ng/ml. In late lactation, alpha-lactalbumin was approximately 70 ng/ml and beta-lactoglobulin approximately 20 ng/ml. Short-term elevations were found after cessation of milking in both alpha-lactalbumin and beta-lactoglobulin in serum. The concentrations of alpha-lactalbumin and beta-lactoglobulin at second parturition were similar to those at first parturition with no differences found between parity. Both alpha-lactalbumin and beta-lactoglobulin in serum were functionally associated with mammary growth and development. In heifers late in pregnancy, both serum concentrations of alpha-lactalbumin and beta-lactoglobulin were positively correlated with mature equivalent milk and fat yields in the subsequent lactation. Serum beta-lactoglobulin concentrations at 16 wk prepartum in heifers were highly correlated with the sum of first and second lactation milk (r = .60) and fat (r = .60) yields. The potential value of using serum beta-lactoglobulin as an index for prescreening of heifers for lactation potential is discussed.

Bovine placental lactogen stimulates DNA synthesis of bovine mammary tissue maintained in athymic nude mice.

Mammary tissue from five midpregnant heifers was transplanted subcutaneously into ovariectomized athymic mice (eight pieces/mouse). After a recovery period of 19 days, mice were injected daily for 5 days with buffer (50 mM NH4HCO3, pH 7.8) as control, 17 beta-estradiol (1 micrograms) plus progesterone (1 mg). Concurrently with the buffer or steroid hormone injections, mice were injected with bovine placental lactogen (0, 5, or 25 micrograms), bovine prolactin (0, 3.4, or 17.2 micrograms), or bovine growth hormone (0, 3.4, or 17.2 micrograms). All mice were injected with 2-bromo-alpha-ergocryptine (0.1 mg/day). Transplanted bovine mammary tissue was incubated for 4 hr in minimum essential medium containing 1 mu Ci/ml [3H]TdR. Two pieces were processed for autoradiography and the others were used for DNA assay and total [3H]TdR uptake. Bovine placental lactogen, prolactin, and growth hormone each increased [3H]TdR incorporation into DNA in a linear, dose-response manner. Addition of ovarian steroids to bPL resulted in a significant increase over protein hormones alone. Autoradiographic analysis indicated that the observed differences in DNA synthesis were due to hormonal effects on epithelial, rather than stromal, DNA synthesis. These results provide the first evidence of a mammogenic role of bovine placental lactogen.

Alteration of milk composition using molecular genetics.

Advances in genetic technology have made it possible to consider making substantial changes either in the composition of milk or in the production of entirely new products in milk. The technological capabilities that have given rise to the introduction and expression of new genes in animals are discussed. Examples are given of transgenic animals that express foreign proteins in their milk. Advantages of the mammary synthesis of proteins are discussed and potential alterations of milk composition and scenarios for introduction of new proteins are considered. Technological capabilities that either currently exist or are being developed are discussed along with the requirements for making it feasible to utilize the technology on a broad scale in dairy cattle.

Strategy for selection of internal, low redundancy probes for identification of specific cDNA.

Internal peptide fragments containing tryptophan residues are useful predictors of coding sequence for selection of restriction enzyme fragments or synthetic oligonucleotides to use in isolation of a cDNA or genomic clone. We describe a strategy to identify such fragments which uses an on-line photodiode array spectrophotometric analysis of tryptic fragment elution from an HPLC system to select peptides for amino acid sequence analysis. We applied this information to the identification and subsequent isolation of a cDNA corresponding to bovine placental lactogen from a bovine placental cDNA library which contains numerous closely related gene products.

Bovine placental lactogen: molecular cloning and protein structure.

The bovine placenta secretes at least one hormone with prolactin-like and placental growth-hormone-like activity. The cDNA for bovine placental lactogen was isolated from a bovine fetal cDNA library by virtue of its nucleotide sequence homology to bovine prolactin (70%) and identified as such from amino acid sequence obtained from the amino terminus and internal tryptic fragments from the isolated hormone. The cDNA predicts a preprohormone of 236 amino acids, with a signal peptide of 36 amino acids. A single consensus site for N-glycosylation marks a probable site of carbohydrate addition. The encoded hormone is quite distinct from the pituitary hormones, as well as the primate and rodent placental lactogens and other predicted bovine placental hormones. It is 51% similar to bovine prolactin in amino acid sequence, 30% similar to the protein predicted by bovine prolactin-related cDNA I, about 30% similar to the rodent predicted placental hormones, and only about 20% similar to human placental lactogen and bovine growth hormone. Despite its greater similarity to bovine prolactin, sequence homology in the region of 5' flanking sequences and first exon to bovine prolactin-related cDNA I suggests that bovine placental lactogen may share a common evolutionary origin with this other placentally expressed member of the prolactin gene family.

Characterization of bovine placental lactogen as a glycoprotein with N-linked and O-linked carbohydrate side chains.

In a previous report we showed that purified bovine placental lactogen (bPL) exists in two isoforms in the 31,000-33,000 Mr range, each with at least five isoelectric variants differing in approximately 2 orders of magnitude in isoelectric points (pI) 4-6. The multiple isoelectric variants are unique to the bovine hormone. In an effort to determine the nature of these variants endo- and exoglycohydrolase digestions were conducted to determine if this hormone was glycosylated. Analysis of peptide/N-glycosidase F and endoglycosidase F digests of radioiodinated bPL on one-dimensional gel electrophoresis showed a Mr decrease from 31,000 to 24,000 and 33,000 to 26,000 for the two isoforms. Digestion with a mixture of neuraminidase plus mixed exoglycosidases resulted in a Mr decrease of 4,000. Digestion with neuraminidase resulted in a Mr decrease of 2,000. Further analysis of peptide/N-glycosidase F- and neuraminidase-treated bPL by two-dimensional gel electrophoresis showed the isoelectric variants shifted from pI 4.4-6.3 to 4.9-8.0. The sialic acid residues on the N-linkage are responsible for the pronounced acidic character of bPL, but do not account for the residual charge heterogeneity as the different isoelectric variants persist after sialic acid removal. The apparent Mr of the protein after removal of N-linked carbohydrate residues is similar to that of PRL and GH. These enzymatic digestion results demonstrate the presence of N-linked complex oligosaccharide residues attached to the beta-amide group of an asparagine residue. Analyses of the sugar content of the molecule were consistent with the presence of one biantennary N-linked and two O-linked carbohydrate chains.

Prolactin, estradiol, and progesterone changes in paretic and nonparetic cows during the periparturient period.

We studied the relationship of serum prolactin, estradiol-17 beta, and progesterone concentrations to plasma calcium, phosphorus, and free hydroxyproline concentrations, as well as to dry matter intake, in 14 aged dairy cows (mean of 4.5 parities), 7 of which became paretic, from 28 days before to 4 days after calving. Plasma calcium and phosphorus concentrations and dry matter intake decreased more at parturition in paretic cows than in nonparetic cows. Prolactin concentrations were not different between paretic and nonparetic cows, but were variable. Concentrations of estradiol were higher in paretic cows from 15 to 5 days before parturition, whereas hydroxyproline concentration was lower in paretic cows on days 10 through 3 before parturition. Progesterone concentration was lower in paretic cows and decreased earlier at parturition, compared with that in nonparetic cows. The findings suggested that high estradiol concentrations in late pregnancy inhibit bone resorption and predispose aged cows to parturient paresis. The earlier decrease in progesterone concentration at parturition and lower concentrations throughout late pregnancy might have contributed to the greater inappetence in paretic cows at parturition. The importance of prolactin in the pathogenesis of parturient paresis is not clear.

The concentration of bovine placental lactogen and the incidence of different forms in fetal cotyledons and in fetal serum.

The concentration of bovine placental lactogen (bPL) was determined in fetal placentomes, allantoic fluid, amniotic fluid, maternal and fetal plasma throughout pregnancy. In addition, chromatofocusing chromatography was used to separate the different forms of bPL found both in fetal serum and in placental homogenates in order to determine whether the different forms that have been reported to exist in the cotyledon are also found in the fetal circulation. Reproductive tracts were collected from cows between 109 and 247 days of pregnancy. The concentration of bPL in the fetal cotyledonary tissue was measured by both radioreceptor assay and radioimmunoassay, both assays showed that the concentration of bPL in the fetal portion of the placentomes remained constant throughout the period of pregnancy tested. The mass of the placenta increased approximately 10-fold during the period of study but the concentration of bPL in the maternal plasma was low (0.9 +/- 0.1 ng/ml) at all stages of pregnancy tested. The mean concentration of bPL (Mean +/- S.E.M.) in amniotic and allantoic fluid was 0.4 +/- 0.1 and 1.2 +/- 0.2 ng/ml respectively. Fetal blood contained the highest concentrations of bPL, from 11.6 to 18.4 ng/ml, and the concentration tended to decrease with advancing gestation (slope = 0.07, P = 0.001). Several forms of bPL were found in the fetal circulation; however, a higher percentage of forms with more acidic isoelectric points were found in the fetal serum than in placental homogenates. These results suggest that either some forms of bPL are more stable or that the hormone isolated from placental tissue is not representative of the final secreted product.