Natural disease resistance refers to the inherent capacity of an animal to resist disease when exposed to pathogens, without prior exposure or immunization (Adams and Templeton, 1998; Caron et al., 2004). Previous studies conducted in our laboratory resulted in the identification of a Black Angus herd sire which was confirmed to be genetically resistant to in vitro and in vivo challenge virulent with Brucella abortus (Qureshi et al., 1996; Adams et al., 1999). Brucellosis is an important zoonotic disease of mammals caused by Brucella spp. and is characterized by its ability to cause abortions, birth of weak or nonviable offspring, and infertility in males and females.
The disease-resistant sire was utilized to initiate breeding studies, determine if natural resistance to bovine brucellosis was heritable, and to identify and characterize the genes involved. Bovine Solute Carrier 11A1 (SLC11A1) also known as natural resistance associated macrophage protein gene 1 (NRAMP 1), which had been previously identified as Lsh/Ity/Bcg gene in mice and humans (Mock et al., 1990, Vidal et al., 1993, Cellier et al., 1994), is conserved on Bos taurus autosome, BTA 2 (Beever et al., 1994; Feng et al., 1996, Barthel et al., 2001) and was identified as the major candidate gene for controlling natural resistance and susceptibility to bovine brucellosis in cattle (Harmon et al., 1985; Harmon et al., 1989; Qureshi et al., 1996).
Unfortunately, our foundation sire died in 1996. In addition, all frozen semen derived from this bull was accidentally destroyed due to improper maintenance in a liquid nitrogen (LN2) storage tank. As a result, the ability to conserve and propagate the genome of this unique animal and produce additional offspring was lost. Given our previous success with cloning cattle by somatic cell nuclear transfer (Hill et al., 2000), we decided to employ this technique to rescue the genotype of this bull using cryopreserved fibroblasts.
MATERIALS AND METHODS
All procedures involving animals were approved by the Texas A&M University Institutional Animal Use and Care Committee operated under the guidelines and certification of the Association for Assessment and Accreditation of Laboratory Animal Care, USDA, and Texas Animal Health Commission.
Production of Cloned Bull
Adult fibroblast cell lines were established in 1985 from an ear punch obtained from the disease-resistant bull (referred to as Bull 86). These were expanded in culture using medium composed of Dulbecco Modified Eagle medium (DMEM; Gibco, Life Technologies, Grand Island, NY) supplemented with 10% fetal calf serum (FCS; Gibco), then frozen and stored in LN2. In 2000, (approximately 15 yr later) the cell lines were thawed and plated into 4-well dishes (Nunc Inc., Naperville, IL) containing DMEM/F-12 medium (Gibco) supplemented with 10% FCS + 1% penicillin/streptomycin (10,000 U/mL of penicillin G, 10,000 μg/mL of streptomycin, Gibco) and maintained for 3 to 5 d. For recombination with the enucleated oocytes, the cultured donor cells were treated with trypsin-EDTA solution (Sigma, St. Louis, MO) for less than 1 min with gentle pipetting. After the addition of 3 mL of HEPES-buffered Tissue Culture Medium (TCM 199, Gibco) supplemented with 10% FCS, the donor cells were washed by centrifugation (3 min, 200 × g), then resuspended in the medium.
Bovine cumulus cell oocyte complexes were collected from abattoir-derived ovaries and matured for 20 h in TCM 199 supplemented with 10% FCS and 1% penicillin/streptomycin and, on a per milliliter basis, 0.1 units of FSH (Sioux Biochem, Sioux City, IA), 0.1 units of LH (Sioux Biochem), 1 μg of estradiol (Sigma), 28 μg of pyruvate (Sigma), and 0.05 μg of epidermal growth factor (EGF, Sigma). After in vitro maturation, expanded cumulus-oocyte complexes were denuded by vortexing for 3 min in 0.1% hyaluronidase (Sigma) in Tyrodes Lactate, HEPES buffered medium (TL-HEPES, Gibco), then washed and placed in TCM 199 with 10% FCS.
Oocytes were enucleated at 21 h postmaturation. Before enucleation, they were placed for 10 min in HEPES-buffered TCM 199 supplemented with 10% FCS and containing (per mL) 5.0 μg of cytochalasin B (Sigma) and 5 μg of Hoechst 33342 (Sigma). All oocytes were carefully selected for the presence of the first polar body and a homogeneous cytoplasm. Enucleation was performed using a beveled 18- to 20-μm o.d. glass pipette mounted on Narishige micromanipulators (Medical Systems Corp., Great Neck, NY) and a Zeiss Microscope (Axiovert 135, Zeiss, Germany). Only oocytes in which the removal of the polar body and the metaphase chromosomes was confirmed by observation under UV light were used. After trypsin/EDTA treatment of cultured donor cell lines, fibroblasts of a median (18- to 20-μm) size and a morphologically round, smooth shape were combined with enucleated oocytes. The oocyte-fibro-blast couplets were then placed into fusion medium consisting of 275 mM mannitol (Sigma), 0.1 mM CaCl2 (Sigma), and 0.1 mM MgSO4 (Sigma).
After equilibration, the couplets were transferred into a 1-mm fusion chamber containing fusion medium, and fused with two 25-μsec, 2.3-kV/cm, direct current pulses delivered by a BTX Electrocell Manipulator 200 (BTX, San Diego, CA). Couplets were then moved to TCM 199 supplemented with 10% FCS and containing 5.0 μg of cytochalasin B (Sigma)/mL and cultured for 1 h before being transferred to cytochalasin B-free medium for an additional 1 h. Fusion was then evaluated, and successfully fused embryos were selected and subjected to an activation treatment.
Embryo activation was performed by a 4-min incubation in 5 μM ionomycin (Calbiochem, San Diego, CA) followed by 4 min in TL-HEPES with 30 mg of BSA (Sigma)/mL. Embryos were then washed twice in TL-HEPES supplemented with 3 mg/mL of BSA. Successfully fused and activated embryos were then placed in a culture well (Nunc) containing 500 μL of TCM 199 supplemented with 10% FCS, 10 μg/mL of CHX (Sigma), and 5 μg/mL of cytochalasin B (Sigma) for 5 h.
After activation, cloned embryos were cultured in G1.2/G2.2 media (Colorado Center for Reproductive Medicine, Englewood, CO) for 7 d as previously described (Gardner et al., 1994). Embryo development was assessed 7 d after cell fusion. In some cases, embryos that had developed to the blastocyst stage were nonsurgically transferred into synchronized recipient cows. Pregnancy status of cows receiving embryos was assessed by transrectal ultrasonography (Aloka 500, 5-MHz transducer, Aloka Co., Tokyo, Japan) at 40 d after nuclear transfer and rechecked on a routine basis.
To confirm that the bull calf was genetically identical to the original cell donor, genotyping was conducted as described previously (Schnabel et al., 2000). In brief, genomic DNA was isolated from white blood cells using the Super Quick-Gene DNA isolation kit (Analytical Genetic Testing Center Inc., Denver, CO). Polymerase chain reaction was utilized, and the products were separated on an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) and sized relative to the internal size standard, Mapmarker low (Bioventures, Murfreesboro, TN). Fluorescent signals from the dye-labeled microsatellites were detected using GeneScan 3.1 software (Applied Biosystems). Alleles were assigned relative to the allelic ladder. Twelve microsatellite markers were utilized for the analysis.
In Vitro Phenotyping for Disease Resistance
The original purebred Angus bull (Bull 86) and his clone (Bull 862) were sequentially studied in 1992 and 2003, respectively. An in vitro, bacterial-killing (expressed as percentage reduction in intracellular survival) assay was employed to phenotype and compare Bull 86, Bull 862, and control animals for disease resistance (Campbell et al., 1994). Bull 86 was characterized as resistant to conjunctival challenge with 107 cfu of live B. abortus S2308 and the ability of his peripheral blood monocyte-derived macrophages to control intra-cellular proliferation of B. abortus (Campbell et al., 1994; Qureshi et al., 1996; Barthel et al., 2001).
To perform the assay, venous blood was collected by aseptic venipuncture into 7.5-mL acid-citrate-dextrose (ACD, Sigma). The blood was diluted with an equal volume of PBS, pH 7.39, containing 13 mM sodium citrate (PBS-C, Gibco). Mononuclear cells were collected by separation of the diluted blood on Percoll (Pharmacia LKB, Uppsala, Sweden) cushions (specific gravity 1.077) by centrifugation at 1,000 × g for 30 min in polypropylene centrifuge tubes. The interface cells, consisting of monocytes, lymphocytes, platelets, and rarely, basophils, were transferred to a clean polypropylene centrifuge tube using disposable polypropylene transfer pipettes and washed 3 times by low speed centrifugation (200 × g) in cold PBS-C to remove platelets and any contaminating Percoll particles. After washing, the cells were suspended at 5 × 106 cells/mL in Roswell Park Memorial Institute (RPMI-1640) medium (Gibco) supplemented with 4 mM
The cells were transferred in 5-mL aliquots to 50-mL Teflon Erlenmeyer flasks (Nalge Company, Rochester, NY) and incubated for 2 h at 38°C in a humidified atmosphere of air with 5% CO2 to allow adherence of monocytes. Nonadherent cells were removed by agitation of the flasks, followed by transfer of the supernatant to a clean 50-mL centrifuge tube. The nonadherent cells were enumerated and used for culture of antigen-responsive lymphocytes for additional studies. Five milliliters of supplemented RPMI-1640 with 12.5% fresh autologous serum were added to each flask, and the flasks were incubated at 38°C in 5% CO2 in air for an additional 48 h, at which time any further nonadherent cells were removed and discarded. The cells were cultured for 10 d, with medium changes at 5 to 7 d to allow maturation to macrophages before use in the assays. All experiments were performed on cells in culture for 10 to 28 d.
The bacterial strains used in this assay were B. abortus ATCC Strain 2308, Mycobacterium bovis BCG Montreal Strain 9003, and Salmonella dublin ATCC Strain 5631. B. abortus and S. dublin were grown on tryptose soy agar (TSA) media plates, whereas M. bovis BCG was grown on Middlebrook 7H10 media plates (BBL, Becton Dickinson Microbiological Systems, Cock-eysville, MD). Bacterial stocks were stored at −70°C as a suspension of 107 cfu/mL in RPMI-1640 supplemented with 15% FCS containing amino acids,
Monocyte-derived macrophages were harvested by chilling the culture flasks on ice for 10 to 15 min, followed by agitation and pipetting to dislodge adherent cells. The harvested cells were enumerated and resuspended at a cell density of 1 million/mL in supplemented RPMI-1640 with 12.5% fresh autologous serum. Cell suspensions (10 μL, 10,000 cells/mL) were transferred to triplicate wells of tissue culture, 60-well, HL-A plates (Nunc) at 750 × g for 5 min and incubated at 38 °C in 5% CO2 in air for 12 to 16 h prior to assay. To prevent desiccation of the well contents, 50 to 100 μL of sterile, distilled water were pooled in each corner of the culture plate.
After the 12- to 16-h incubation, the well contents were aspirated, and 5 μL of a suspension of B. abortus S2308 (10 million cells/mL) was added to each well. The plates were centrifuged at 750 × g for 5 min and incubated at 38°C in a humidified atmosphere supplemented with 5% CO2. After a 30-min incubation to allow bacteria-cell interaction, 10 μL of a 37.5 μg/mL solution of gentamycin sulfate (Gibco) were added to each well to a final concentration of 25 μg/mL, with further incubation for 1 h. As a control for the killing of extracellular bacteria, 0.5 mL of bacterial suspension was added to 1 mL of antibiotic solution and incubated concomitantly with the culture plates. This kept bacteria and antibiotic in the same relative concentrations as in the test wells and provided a large enough sample to facilitate handling. An additional control for adequacy of bacterial washing included a tube containing 0.5 mL of bacterial suspension and 1.0 mL of RPMI-1640.
After the antibiotic treatment, all wells were washed 4 times with fresh, unsupplemented medium, followed by lysis of the macrophages by addition of 10 μL of 0.5% Tween-20 (Sigma) in sterile, distilled water. Well contents were serially diluted in sterile, distilled water, and 100 μL of the dilutions was plated on TSA for enumeration of colony-forming units. The contents of the antibiotic treatment control and bacterial wash control tubes were washed 3 times with distilled water, resuspended to 2 mL of total volume, and 100 μL was plated on TSA. This corresponded in concentration to the first dilution tube after well harvest. At 12 h, the wells of the duplicate culture plate were similarly harvested. The percent survival of bacteria was determined for triplicate samples over the 12-h incubation period. Because the populations of cattle were specifically chosen from or included members of pedigreed families based on the response to a challenge infection with live B. abortus, the data were not normally distributed. Data were analyzed for significance by the nonparametric Mann-Whitney U-test.
Of 493 1-cell (fused) cloned embryos, 223 (45%) developed to the blastocyst stage after in vitro culture. Of these, 39 expanded or hatching blastocysts were selected and transferred to 20 synchronized recipient cows (all but 1 recipient received 2 embryos at the time of transfer). Ten recipients (50%) were diagnosed pregnant at 40 d by ultrasonography. Nine of 10 pregnancies spontaneously aborted (4 at d 44 to 45, 4 at d 68 to 92, and 1 at d 214 of gestation, respectively). One survived to term resulting in a live bull calf at d 283 of gestation. The bull calf, which was delivered by Caesarian section after induction with i.v. glucocorticoid dexamethasone (Azium, Schering-Plough Inc., Kenilworth, NJ) treatment (20 mL of 2 mg/mL) in order to facilitate maturation of the fetal respiratory system, appeared normal and healthy at birth. The genotype of the cloned bull matched the original donor DNA at all 12 markers, indicating it was a clone of the original cell donor. The cloned bull (Bull 862) is now 4.5 yr old. A picture of Bull 862 at 2 yr of age is provided in Figure 1.
The results of the in vitro killing assays performed in 2003 demonstrated that macrophages cultured from Bull 862 (cloned bull) reduced bacterial survival of B. abortus S2308 (59.6%), M. bovis BCG (64.0%), and S. dublin (73.3%). These results were similar when compared with macrophages cultured from Bull 86 for reduction of bacterial survival of B. abortus S2308 (61.0%), M. bovis BCG (66.3%), and S. dublin (76.6%) performed in 1993 using the identical procedures, instrumentation, and technical personnel (Table 1). Thus the in vitro microbial killing profiles of the macrophages of Bull 86 and Bull 862 were virtually identical. Clearly, Bull 86 and Bull 862 had significantly greater pathogen killing capacities as compared with previously published reports for the pathogen survival capacities of susceptible cattle respectively for B. abortus S2308 (120 to 180%), M. bovis BCG (125 to 225%), and S. dublin (150 to 425%) (Harmon et al., 1985;; Campbell et al., 1994; Qureshi et al., 1996).
In this study, we demonstrated that long-term cryopreserved somatic cells (15 yr) can be successfully reprogrammed and result in the live birth of a cloned offspring through nuclear transfer technology. The cell donor was a deceased purebred black Angus bull registered with the American Black Angus Association and previously recognized as one that had inherited the SLC11A1 gene, which imparts resistance against bovine brucellosis (Feng et al., 1996). Due to advances with animal cloning technology, we attempted to clone this bull by using frozen-thawed cells that had been collected from ear skin in 1985. As far as we know, this is the first cloned bull produced from a frozen-thawed tissue sample after long-term storage for 15 yr in LN2. The cells used for cloning in this study were frozen more than 10 yr before the first report of successful cloning from adult cells (Wilmut et al., 1997) and at a time when cloning animals using somatic cells was thought to be biologically impossible. The relevance of this lies in the fact that more than a dozen mammalian species have now been cloned by somatic cell nuclear transfer. Although difficult to predict, there could easily be thousands of potentially valuable animal genomes stored in the form of somatic cells in LN2 over the last several decades that could be thawed and utilized to recover valuable genotypes.
Previous studies have demonstrated that cloned offspring do not always exhibit phenotypes identical to the original cell (nucleus) donor (Shin et al., 2002; Archer et al., 2003). We and others have shown this phenomenon likely due to inadequate reprogramming of the donor nucleus after nuclear transplantation, resulting in abnormal epigenetic regulation of gene expression (De Sousa et al., 1999; Humpherys et al., 2002). Therefore, we were not certain whether Bull 862 would exhibit a disease-resistant phenotype. In vitro bacterial killing assays were performed and demonstrated that (similar to the original Bull 86), the cloned bull (Bull 862) is resistant to brucellosis, indicating that the genes affording disease resistance were not affected by the nuclear transfer procedure and are functioning properly. Had Bull 862 not exhibited a disease-resistant phenotype as a result of inadequate nuclear reprogramming, it is unlikely that this phenotype would be passed on to his offspring. Previous studies in mice have clearly demonstrated that although cloned animals may exhibit abnormal gene expression patterns, this problem is corrected when these animals are used for breeding, and the offspring of clones exhibit normal gene expression (Tamashiro et al., 2002). To date, we have not produced any offspring using semen collected from Bull 862, but based on the in vitro data, we predict all his offspring will exhibit the genotype and phenotype of the original Bull 86. Furthermore, we reproduced a desired genetically identical animal that can allow us further study of the genetic background related to disease resistance. This study provides direct evidence for the feasibility of long-term conservation by cryopreservation and rescue of genetic materials when combined with animal cloning technologies.
|Replicates||——— Clone 862 ———|
|——— Original 86 ———|
|862 vs. 86||P = 0.7||P = 1||P = 1|