Targeted disruption of the porcine immunoglobulin kappa light chain locus.

Inactivation of the endogenous pig immunoglobulin (Ig) loci, and replacement with their human counterparts, would produce animals that could alleviate both the supply and specificity issues of therapeutic human polyclonal antibodies (PAbs). Platform genetics are being developed in pigs that have all endogenous Ig loci inactivated and replaced by human counterparts, in order to address this unmet clinical need. This report describes the deletion of the porcine kappa (κ) light chain constant (Cκ) region in pig primary fetal fibroblasts (PPFFs) using gene targeting technology, and the generation of live animals from these cells via somatic cell nuclear transfer (SCNT) cloning. There are only two other targeted loci previously published in swine, and this is the first report of a targeted disruption of an Ig light chain locus in a livestock species. Pigs with one targeted Cκ allele (heterozygous knockout or ±) were bred together to generate Cκ homozygous knockout (-/-) animals. Peripheral blood mononuclear cells (PBMCs) and mesenteric lymph nodes (MLNs) from Cκ -/- pigs were devoid of κ-containing Igs. Furthermore, there was an increase in lambda (λ) light chain expression when compared to that of wild-type littermates (Cκ +/+). Targeted inactivation of the Ig heavy chain locus has also been achieved and work is underway to inactivate the pig lambda light chain locus.

Generation of antibody- and B cell-deficient pigs by targeted disruption of the J-region gene segment of the heavy chain locus.

A poly(A)-trap gene targeting strategy was used to disrupt the single functional heavy chain (HC) joining region (J(H)) of swine in primary fibroblasts. Genetically modified piglets were then generated via somatic cell nuclear transfer (SCNT) and bred to yield litters comprising J(H) wild-type littermate (+/+), J(H) heterozygous knockout (±) and J(H) homozygous knockout (-/-) piglets in the expected Mendelian ratio of 1:2:1. There are only two other targeted loci previously published in swine, and this is the first successful poly(A)-trap strategy ever published in a livestock species. In either blood or secondary lymphoid tissues, flow cytometry, RT-PCR and ELISA detected no circulating IgM(+) B cells, and no transcription or secretion of immunoglobulin (Ig) isotypes, respectively in J(H) -/- pigs. Histochemical and immunohistochemical (IHC) studies failed to detect lymph node (LN) follicles or CD79α(+) B cells, respectively in J(H) -/- pigs. T cell receptor (TCR)(ß) transcription and T cells were detected in J(H) -/- pigs. When reared conventionally, J(H) -/- pigs succumbed to bacterial infections after weaning. These antibody (Ab)- and B cell-deficient pigs have significant value as models for both veterinary and human research to discriminate cellular and humoral protective immunity to infectious agents. Thus, these pigs may aid in vaccine development for infectious agents such as the pandemic porcine reproductive and respiratory syndrome virus (PRRSV) and H1N1 swine flu. These pigs are also a first significant step towards generating a pig that expresses fully human, antigen-specific polyclonal Ab to target numerous incurable infectious diseases with high unmet clinical need.

Mammary epithelial proliferation and estrogen receptor alpha expression in prepubertal heifers: effects of ovariectomy and growth hormone.

The objectives of this study were to determine the effects of ovariectomy and growth hormone on mammary epithelial cell proliferation and estrogen receptor alpha (ER alpha) expression within the bovine mammary gland. Two experiments were performed. In the first experiment, eight Holstein heifer calves aged between 1 and 3 mo were ovariectomized, while six calves served as controls. At 6 mo of age, calves were treated with bromodeoxyuridine (BrdU) to label proliferating cells and sacrificed 2 h later. Coinciding with reduced mammary mass (304 +/- 25 vs. 130 +/- 21 g), proliferation of mammary epithelial cells was significantly lower in ovariectomized heifers compared to control heifers (2.24 vs. 0.25%). ER alpha expression was restricted to mammary epithelial cells and was not observed within intra-lobular stroma of parenchymal tissue. The proportion of ER alpha positive cells was significantly higher in ovariectomized heifers than in controls (36.1% +/- 2.2 vs. 46.7% +/- 2.4). In the second experiment, mammary biopsies were taken from five 6-mo-old heifers, immediately preceding and 7 d following a single injection of bovine growth hormone. Mammary epithelial cell proliferation (assessed by incorporation of 3H-thymidine) was increased by growth hormone. The proportion of ER alpha positive mammary epithelial cells was not increased by growth hormone. In conclusion, reduced mammary epithelial cell proliferation following ovariectomy was associated with an increase in ER alpha expression, whereas increased proliferation caused by bovine growth hormone was not associated with changes in the proportion of ER alpha positive cells.

Interactions between the ovary and the local IGF-I axis modulate mammary development in prepubertal heifers.

The objective was to determine the effects of ovariectomy and epithelial-stromal interactions on mammary development and local expression of IGF-I and IGF-binding protein (IGFBP) mRNA in prepubertal heifers. An epithelium-free ('cleared') fat pad (CFP) was prepared in two glands in each of 14 Holstein heifers, aged 1-3 Months. Eight of the calves were also ovariectomized. Serum concentrations of GH, IGF-I and prolactin were not affected by ovariectomy. At 6 Months of age, calves were killed to provide mammary samples of parenchyma, CFP and intact fat pad (MFP). Total mammary mass was reduced in ovariectomized calves (130+/-21 g vs 304+/- 25 g; P<0.001), and in several cases parenchymal tIssue was essentially absent. Uterus weight was also reduced by ovariectomy (14.5+/-3.8 g vs 30.4+/-4.5 g; P<0.05). In support of our hypothesis that local IGF-I mediates prepubertal mammary development, mRNA expression of IGF-I was lower in ovariectomized than in control calves (62.1+/-7.8 vs 91.6+/-7.8 arbitrary units; P<0.05). Specific binding of IGF-I to mammary parenchymal microsomes was also reduced by ovariectomy (377+/-142 vs 868+/-82 c.p.m.; P<0.01), suggesting decreased sensitivity to IGF-I. Expression of IGFBP-3 and IGFBP-5 mRNA were not influenced by ovariectomy. Expression of IGF-I, IGFBP-3 and IGFBP-5 mRNA did not differ between CFP and MFP, suggesting that expression of these factors was not influenced by interactions between stroma and developing epithelium. Overall, the data suggested that interactions between the ovary and the local IGF-I axis act to optimize the availability and effectiveness of IGF-I within the gland to stimulate mammary growth.

Preimplantation screening for transgenesis using an embryonic specific promoter and green fluorescent protein.

We report enrichment in the efficiency of generating mice transgenic for expression of a human protein in their milk using GFP-mediated preimplantation screening. The transgene array consisted of a functional gene (human alpha-1 antitrypsin under the control of the ovine BLG promoter) linked 5' to a reporter gene (GFP under the control of the murine Oct-4 promoter). GFP expression was detected in blastocysts by fluorescence microscopy and green and nongreen embryos were transferred to recipients in separate groups. In the first experiment, of seven pups that resulted from the transfer of blastocysts expressing GFP, five (71%) were transgenic. The experiment was repeated and of 12 pups that resulted from transfer of GFP-expressing blastocysts, 11 were transgenic (92%). The presence of the reporter cassette used for preimplantation screening did not affect the expression level of alpha-1-antitrypsin in the milk of the transgenic mice. In addition, in a related experiment wherein the GFP reporter gene was co-injected with a second mammary-specific transgene, pINC, no effect on transgene expression was observed. For mice transgenic for the mammary-specific gene alone, expression levels for four different lines were 192, 197, 382, and 415 microg/mL. For mice transgenic for both the mammary-specific transgene and the Oct4-GFP reporter cassette, expression levels for seven different lines were 282, 321, 468, 497, 499, 516, and 806 microg/mL.