Search

Considerable progress has been made in methods for production of transgenic livestock; beginning with pronuclear microinjection over 20 years ago. New methods, including the use of viral vectors, sperm-mediated gene transfer and somatic cell cloning, have overcome many of the limitations of pronuclear microinjection. It is now possible to not only readily make simple insertional genetic modifications, but also to accomplish, more complex, homozygous gene targeting and artificial chromosome transfer in livestock.

Prion diseases are caused by propagation of misfolded forms of the normal cellular prion protein PrP(C), such as PrP(BSE) in bovine spongiform encephalopathy (BSE) in cattle and PrP(CJD) in Creutzfeldt-Jakob disease (CJD) in humans. Disruption of PrP(C) expression in mice, a species that does not naturally contract prion diseases, results in no apparent developmental abnormalities. However, the impact of ablating PrP(C) function in natural host species of prion diseases is unknown. Here we report the generation and characterization of PrP(C)-deficient cattle produced by a sequential gene-targeting system. At over 20 months of age, the cattle are clinically, physiologically, histopathologically, immunologically and reproductively normal. Brain tissue homogenates are resistant to prion propagation in vitro as assessed by protein misfolding cyclic amplification. PrP(C)-deficient cattle may be a useful model for prion research and could provide industrial bovine products free of prion proteins.

We have developed a novel system for remodeling mammalian somatic nuclei in vitro prior to cloning by nuclear transplantation. The system involves permeabilization of the donor cell and chromatin condensation in a mitotic cell extract to promote removal of nuclear factors solubilized during chromosome condensation. The condensed chromosomes are transferred into enucleated oocytes prior to activation. Unlike nuclei of nuclear transplant embryos, nuclei of chromatin transplant embryos exhibit a pattern of markers closely resembling that of normal embryos. Healthy calves were produced by chromatin transfer. Compared with nuclear transfer, chromatin transfer shows a trend toward greater survival of cloned calves up to at least 1 mo after birth. This is the first successful demonstration of a method for directly manipulating the somatic donor chromatin prior to transplantation. This procedure should be useful for investigating mechanisms of nuclear reprogramming and for making improvements in the efficiency of mammalian cloning.

Human polyclonal antibodies (hPABs) are useful therapeutics, but because they are available only from human donors, their supply and application is limited. To address this need, we prepared a human artificial chromosome (HAC) vector containing the entire unrearranged sequences of the human immunoglobulin (hIg) heavy-chain (H) and lambda (lambda) light-chain loci. The HAC vector was introduced into bovine primary fetal fibroblasts using a microcell-mediated chromosome transfer (MMCT) approach. Primary selection was carried out, and the cells were used to produce cloned bovine fetuses. Secondary selection was done on the regenerated fetal cell lines, which were then used to produce four healthy transchromosomic (Tc) calves. The HAC was retained at a high rate (78-100% of cells) in calves and the hIg loci underwent rearrangement and expressed diversified transcripts. Human immunoglobulin proteins were detected in the blood of newborn calves. The production of Tc calves is an important step in the development of a system for producing therapeutic hPABs.

Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs) in transchromosomic (Tc) cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin (Ig) heavy-chain (hIGH), kappa-chain (hIGK), and lambda-chain (hIGL) germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM-/-, bIGHML1-/-; double knockouts or DKO). However, because endogenous bovine immunoglobulin light chain loci are still intact, the light chains are produced both from the hIGK and hIGL genomic loci on the HAC and from the endogenous bovine kappa-chain (bIGK) and lambda-chain (bIGL) genomic loci, resulting in the production of fully hIgGs (both Ig heavy-chains and light-chains are of human origin: hIgG/hIgκ or hIgG/hIgλ) and chimeric hIgGs (Ig heavy-chains are of human origin while the Ig light-chains are of bovine origin: hIgG/bIgκ or hIgG/bIgλ). To improve fully hIgG production in Tc cattle, we here report the deletion of the entire bIGL joining (J) and constant (C) gene cluster (bIGLJ1-IGLC1 to bIGLJ5-IGLC5) by employing Cre/loxP mediated site-specific chromosome recombination and the production of triple knockout (bIGHM-/-, bIGHML1-/- and bIGL-/-; TKO) Tc cattle. We further demonstrate that bIGL cluster deletion greatly improves fully hIgGs production in the sera of TKO Tc cattle, with 51.3% fully hIgGs (hIgG/hIgκ plus hIgG/hIgλ).

Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT) is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT). Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively) have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF) than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively). However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research.

If the full potential of chromatin transfer (CT) technology is to be realized for both animal production and biomedical applications it is imperative that the efficiency of the reprogramming process be improved, and the potential for deleterious development be eliminated. Generation of the first cloned animals from adult somatic cells demonstrated that development is substantially an epigenetic process (Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH, 1997. Viable offspring derived from fetal and adult mammalian cells. Nature. 385(6619): 810–813.). In this study, we provide preliminary evidence that the epigenetic state of the donor cell, may be valuable in assessing potential cloning success. We have measured key indicators of cellular epigenetic state in both serially derived cell populations of the same genetic origin, but differing in epigenomic status, and in a distinct cohort of donor cell populations with diverse genetic origins and epigenomic status. Specifically, the relative abundance of particular histone modifications in donor populations prior to manipulation has been correlated with the measurable variance in reprogramming efficiencies observed following CT, as defined by the number of resulting live births and healthy progeny, and the concomitant incidence of deleterious growth measures (notably the appearance of large offspring syndrome (LOS)). Thus, we suggest that the likely outcome and relative success of cloning may be predictable based on the expression of discriminating histone marks present in the donor cell population before CT. This approach may provide the basis of a prognostic signature for the future evaluation and risk assessment of putative donor cells prior to CT, and thus increase future cloning success and alleviate the incidence of abnormal development.

Antigen-specific human polyclonal antibodies (hpAbs), produced by hyperimmunization, could be useful for treating many human diseases. However, yields from available transgenic mice and transchromosomic (Tc) cattle carrying human immunoglobulin loci are too low for therapeutic applications. We report a Tc bovine system that produces large yields of hpAbs. Tc cattle were generated by transferring a human artificial chromosome vector carrying the entire unrearranged, human immunoglobulin heavy (hIGH) and kappa-light (hIGK) chain loci to bovine fibroblasts in which two endogenous bovine IgH chain loci were inactivated. Plasma from the oldest animal contained >2 g/l of hIgG, paired with either human kappa-light chain (up to approximately 650 microg/ml, fully human) or with bovine kappa- or lambda-light chain (chimeric), with a normal hIgG subclass distribution. Hyperimmunization with anthrax protective antigen triggered a hIgG-mediated humoral immune response comprising a high proportion of antigen-specific hIgG. Purified, fully human and chimeric hIgGs were highly active in an in vitro toxin neutralization assay and protective in an in vivo mouse challenge assay.

Gene targeting is accomplished using embryonic stem cells in the mouse but has been successful, only using primary somatic cells followed by embryonic cloning, in other species. Gene targeting in somatic cells versus embryonic stem cells is a challenge; consequently, there are few reported successes and none include the targeting of transcriptionally silent genes or double targeting to produce homozygotes. Here, we report a sequential gene targeting system for primary fibroblast cells that we used to knock out both alleles of a silent gene, the bovine gene encoding immunoglobulin-mu (IGHM), and produce both heterozygous and homozygous knockout calves. We also carried out sequential knockout targeting of both alleles of a gene that is active in fibroblasts, encoding the bovine prion protein (PRNP), in the same genetic line to produce doubly homozygous knockout fetuses. The sequential gene targeting system we used alleviates the need for germline transmission for complex genetic modifications and should be broadly applicable to gene functional analysis and to biomedical and agricultural applications.

We have developed a novel system for remodeling mammalian somatic nuclei in vitro prior to cloning by nuclear transplantation. The system involves permeabilization of the donor cell and chromatin condensation in a mitotic cell extract to promote removal of nuclear factors solubilized during chromosome condensation. The condensed chromosomes are transferred into enucleated oocytes prior to activation. Unlike nuclei of nuclear transplant embryos, nuclei of chromatin transplant embryos exhibit a pattern of markers closely resembling that of normal embryos. Healthy calves were produced by chromatin transfer. Compared with nuclear transfer, chromatin transfer shows a trend toward greater survival of cloned calves up to at least 1 mo after birth. This is the first successful demonstration of a method for directly manipulating the somatic donor chromatin prior to transplantation. This procedure should be useful for investigating mechanisms of nuclear reprogramming and for making improvements in the efficiency of mammalian cloning.

Artificial chromosome vectors are autonomous, replicating DNA sequences containing a centromere, two telomeres and origins of replication. Artificial chromosomes have been proposed as possible vectors for transferring very large sequences of DNA into animals. Our goal has been to insert the entire human heavy- and light-chain immunoglobulin loci into cattle as a step in developing a production system for large quantities of human therapeutic polyclonal antibodies. A mitotically stable fragment of chromosome 14, containing the human heavy-chain locus, was identified. A chromosome cloning system was used to transfer the human lambda locus from an unstable chromosome 22 fragment to the chromosome 14 fragment to create a human artificial chromosome (HAC) carrying both immunoglobulin loci. The HAC vector was introduced into bovine primary fibroblasts. Selected fibroblast clones were rejuvenated and expanded by producing cloned fetuses. Cloned fetal cells were selected and recloned to produce 21 healthy, transchromosomic (Tc) calves. Four were analyzed and shown to functionally rearrange both heavy- and light-chain human immunoglobulin loci and produce human polyclonal antibodies. These results demonstrate the feasibility of using HAC vectors for production of transgenic livestock. More importantly, Tc cattle containing human immunoglobulin genes may be used to produce novel human polyclonal therapeutics.

Pregnancies obtained via somatic cell nuclear transfer (SCNT) show an increased incidence of pregnancy complications, including poor placental vascularization, hydrops placenta, and abnormal placental morphology. In a preliminary study of hydrops placentas from SCNT clones v. control cow placentas close to term, we observed a thickening of the endometrial epithelium, which appeared pseudostratified; in addition, the subepithelial endometrial vascular bed consisted of large, rare arterioles with thick basement membranes compared with the normal capillary bed observed in control animals. The fetal membranes of hydrops placenta from bovine SCNT clones also exhibited macroscopic morphological differences, which included edema, increased membrane thickness, and a gelatinous appearance. All of these morphological differences were similar to those we have reported for sheep SCNT clones (Palmieri et al. 2007 Placenta 28, 577–584). In the search for biomarkers that may indicate abnormal placental development early in pregnancy, placental tissues were collected on day 40 from pregnancies established after transfer of embryos created through SCNT (n = 17) or IVF (n = 6). Nuclear donor cells used to obtain SCNT embryos were those we have previously found to be highly susceptible to hydrops (from 5 to 19%), whereas the incidence of hydrops in IVF and controls in our laboratories did not exceed 0.5%. A sample of fetal membranes was fixed in 10% buffered formalin for histological analysis; another sample was snap-frozen in liquid nitrogen for analysis of mRNA levels for vascular endothelial growth factor (VEGF) and its receptors (KDR and FLT) by quantitative, real-time RT-PCR. Histological analysis of cell proliferation rate was performed using immunohistochemical localization of proliferating cell nuclear antigen and image analysis software (Image-Pro Plus). There were no differences (P = 0.39) in the rate of cell proliferation of the trophoblast in SCNT v. IVF (29.4 ± 4.9% v. 22.1 ± 3%). However, VEGF mRNA expression was less (P < 0.04) in the fetal membranes of SCNT than IVF on day 40 of pregnancy (0.30 ± 0.04 v. 0.53 ± 0.12, normalized to α-actin mRNA levels). The mRNA expression for KDR and FLT were similar in SCNT and IVF. These data suggest that the lower expression of VEGF, a major placental angiogenic factor, in fetal membranes of early pregnant SCNT fetuses may be an early biomarker for the development of hydrops placenta later in pregnancy. NDAES Proj. ND01727.

Bovine oocytes derived from abattoir origin are mainly used for artificial reproductive techniques in both agricultural and biomedical applications. Regulatory agencies have expressed concern for potential transmission of adventitious viruses by sourcing bovine oocytes from abattoir origin. To evaluate this concern, a viral risk assessment was conducted on batch samples collected from follicular fluid, cumulus cells, oocytes, and Day 8 embryos. These batch samples were collected from ovaries on seven randomly selected days in a 2-week period and they were frozen and stored at –80°C until tested. All samples were tested by 9 Code of Federal Regulations (9CFR) part 113.53c (animal viral testing) at a GLP compliant laboratory (American BioResearch Laboratories, Sevier, TN, USA). The 9CFR viral testing includes bovine viral diarrhea virus (BVDV), bovine parvovirus, bovine adenovirus type 3 and 5, bovine rabies virus, bovine bluetongue virus, bovine respiratory syncytial virus, bovine reovirus, viral cytopathic effect, and hemadsorption on permissive cell cultures. Batch samples were also tested for BVDV and bovine leukemia virus (BLV) by polymerase chain reaction (PCR) and follicular fluids for BVDV antibody neutralization activity at an accredited diagnostic laboratory (Animal Disease Research and Diagnostic Laboratory, SDSU, Brookings, SD, USA). The 9CFR viral testing results on all the batch samples were negative. The BVDV PCR test had a low positive (37.89 cycles) with one follicular fluid batch sample (1/7) and a low positive (37.9 cycles) on all cleaved embryos (7/7). However, BVDV virus isolation was negative for both batch samples by 9CFR testing. The BLV PCR had a positive follicular batch sample (1/7), with all other samples testing being negative. BVD serum neutralization antibody assay demonstrated that all follicular fluid had significant titers of 1:128–1:1024. Although some of the viral particles may have been detected in follicular fluid and cleaved embryos by PCR, none of the batch samples collected were positive for viral growth in this study. The BVDV PCR indicated low levels of BVDV RNA. It is speculated that the positive BVDV PCR results on cleaved embryos could possibly be attributed to the use of irradiated fetal calf serum (contaminated BVDV virus) used in culture media. Follicular fluids also have high titers to BVDV which may have neutralized the virus. These results indicate that virus-free bovine oocytes can be derived from the abattoir. Thus, with appropriately applied quality assurance testing, abattoir-origin oocytes might be used effectively in agricultural and biomedical applications. Table 1. Summary of test results on batch samples from abattoir-derived oocytes

Numerous genetically similar cattle for use in research or teaching can be produced with chromatin transfer technology (Sullivan et al. 2004 Biol. Reprod. 70, 146–153). Miniature cattle can provide advantageous biotechnological models for teaching and study of human and animal diseases. Miniature cattle are approximately one fourth the size of standard cattle and, therefore, represent a potentially less expensive, safer, and more easily managed animal model. Our limited attempts to reproduce miniature cattle via in vivo and in vitro production of embryos resulted in poor response to superovulation and logistical challenges in recovering embryos or oocytes due to the small size of the animals. Thus, the purpose of this study was to evaluate the potential of embryos derived by chromatin transfer from fibroblasts of a miniature cow and cytoplasts from cows of standard size to produce viable offspring after transfer into recipient cows of standard size. The donor of somatic cells was a heifer that weighed 7.7 kg at birth. Chromatin transfer resulted in 19% (82/428) blastocyst formation. A total of 66 cloned blastocysts (65 excellent/good quality, 1 fair quality) were transferred into 26 synchronized recipients. While ultrasound revealed 13 pregnancies prior to 67 days of gestation, only 4 pregnancies of 5 fetuses were maintained beyond 100 days. Parturition was induced with dexamethasone and prostaglandin on Day 286 of gestation. One singleton (12.3 kg) and a set of twins (10.2 and 11.1 kg) were healthy at birth and normal at 1 week of age. Two fully developed singletons, weighing 21.4 and 13.6 kg, died in utero. The latter fetus exhibited a fixed dorsolateral deviation of the neck that complicated delivery despite a caudal obstetrical presentation. No abnormalities were noted in the size or structure of any placenta. Our results indicate that healthy miniature calves can be gestated by recipient cows of standard size after transfer of embryos derived by chromatin transfer. Unfortunately, the fetal wastage, fetal anomalies, and stillbirths observed with standard sized cattle also may occur.

Most isolates of BVDV cause unapparent infections in cultured cells. Fetuses, postnatal animals or fetal bovine serum are possible sources of the virus for cultivated cells used as karyoplasts in cloning. Routine screening by veterinary diagnostic laboratories of 39 fetal fibroblast cell lines used in cloning research had revealed that 15 (39%) were positive for BVDV by various assays including RT-nPCR. As some were valuable transgenic cell lines, a rigorous protocol for evaluation of each line was undertaken to confirm infection with BVDV. A cryopreserved vial of each line was thawed, medium discarded and cells incubated (38.5°C in 5% CO2 and air) through 2 passages (6–10 days) in α-MEM supplemented with 10% equine serum. At the end of the second passage, cells were separated from medium, washed and assayed for presence of BVDV using virus isolation in 2 sequential passages in Madin Darby Bovine Kidney Cells and RT-nPCR. Available lots of fetal bovine serum and medium that had been used to culture the cells also were tested for BVDV. When the virus was detected, the RT-nPCR products were sequenced and compared. Also, an attempt was made to evaluate the earliest available cryopreserved passage of any positive cell lines. Results indicated that just 5 of 39 of the original cells tested (13%) were positive. Since cryopreserved earlier passages of 4 of the cell lines were available, they were assayed with the result that 2 of the 4 were not infected at the earliest passage. Further, BVDV was isolated from one lot of fetal bovine serum that was used to culture one of the cell lines. Sequence analysis verified that only 2 of these 4 cell lines were infected with the same isolate of BVDV, and one isolate was identical to the virus found in the fetal bovine serum used in medium to culture it. The discrepancy between our viral detection and that of the diagnostic laboratories is explained in part by the presumed test protocols. All BVDV-positive cells, as reported by the diagnostic laboratories, were positive by RT-nPCR. We presume that they did not separate medium from cells before assays. Thus, any noninfectious viral RNA that was in the medium (e.g. as would be expected in many lots of irradiated serum) would have been reported positive. The only possible sources for BVDV in these cell lines were the fetuses from which they originated or fetal bovine serum used in medium. Sequence analysis confirmed that serum was the source of viral infection in one line. The likely source of virus for 2 other lines was serum, since they were not infected at earlier passages. The 2 remaining cell lines were positive at the earliest available passages, so the fetuses from which cells were harvested could not be discounted as the source of BVDV. This report highlights the risks of introducing BVDV in embryo technologies and the difficulties that can be encountered in attempting accurate diagnosis of the presence of infectious virus.

Unnoticed infections with bovine viral diarrhea virus (BVDV) can occur in cultured cells used for somatic cell nuclear transfer. Aromatic cationic molecules have exhibited inhibitory activity against in vitro replication of BVDV. The purpose of this research was to evaluate the ability of aromatic cationic compounds to prevent or treat noncytopathic BVDV infections of fetal fibroblast cells. Aromatic compounds tested were 2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan (DB606); 2-(2-benzimidazolyl)-5-[4-(2-imidazolino) phenyl]furan dihydrochloride (DB772); and 2-(1-methyl-2-benzimidazolyl)-5-[4’-(2-imidazolino)-2’-methylphenyl]furan dihydrochloride (DB824). To evaluate prevention of BVDV infections, 10 cell lines in the absence or presence of 7 dilutions of each of the 3 compounds were inoculated with BVDV. The concentrations of BVDV in medium and cell lysates were determined by serial dilution and virus isolation. Samples were obtained 72 hours post-inoculation. Bovine viral diarrhea virus in cell culture medium and cell lysate samples was evaluated by comparison to equivalent samples from control cultures in which no compound was added (percent of control=cell culture infective doses (50%; CCID50) of BVDV in compound sample/CCID50 of BVDV in control sample lacking compound). The viral inhibitory concentrations (99%) of compounds were calculated with JMP software by least-squares regression techniques. Cumulatively, the 99% endpoints for inhibition of viral replication in fetal fibroblast cell lines for the 3 compounds were 0.1μM, 0.007μM and 0.028μM, respectively. To evaluate therapeutic treatment of established BVDV infections, the concentration of BVDV in medium and cell lysates of 2 fetal fibroblast cell lines were evaluated. The cell lines were previously determined to be infected with a genotype 1a strain of BVDV. Samples were obtained during 4 sequential passages in the absence or presence of 0.04μM and 4μM concentrations of DB772 or DB824. Presence of BVDV was determined by reverse transcription nested polymerase chain reaction and virus isolation. While BVDV persisted in cultures supplemented with no aromatic compound or 0.04μM, both DB772 and DB824 effectively cured BVDV infections after 1 passage in 4μM, and cells remained viable. Results indicate that BVDV infections can be effectively prevented or treated in fetal fibroblast cultures. Further research is needed to determine if exposed cells are competent for production of normal embryos via nuclear transfer.