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Published March 12, 2015
Rescuing valuable genomes by animal cloning: A case for natural
disease resistance in cattle1,2
M. E. Westhusin,3 T. Shin, J. W. Templeton, R. C. Burghardt, and L. G. Adams4
Texas A&M University, College of Veterinary Medicine & Biomedical Sciences, College Station, TX
had been established in 1985, cryopreserved, and stored
in liquid nitrogen for future genetic analysis. Therefore,
we decided to utilize these cells for somatic cell nuclear
transfer to attempt the production of a cloned bull and
salvage this valuable genotype. Embryos were produced
by somatic cell nuclear transfer and transferred to 20
recipient cows, 10 of which became pregnant as determined by ultrasound at d 40 of gestation. One calf survived to term. At present, the cloned bull is 4.5 yr old
and appears completely normal as determined by physical examination and blood chemistry. Furthermore, in
vitro assays performed to date indicate this bull is naturally resistant to B. abortus, Mycobacterium bovis, and
Salmonella typhimurium, as was the original genetic
donor.
ABSTRACT: Tissue banking and animal cloning represent a powerful tool for conserving and regenerating
valuable animal genomes. Here we report an example
involving cattle and the rescue of a genome affording
natural disease resistance. During the course of a 2decade study involving the phenotypic and genotypic
analysis for the functional and genetic basis of natural
disease resistance against bovine brucellosis, a foundation sire was identified and confirmed to be genetically
resistant to Brucella abortus. This unique animal was
utilized extensively in numerous animal breeding studies to further characterize the genetic basis for natural
disease resistance. The bull died in 1996 of natural
causes, and no semen was available for AI, resulting
in the loss of this valuable genome. Fibroblast cell lines
Key words: animal cloning, genetic conservation, natural disease resistance
©2007 American Society of Animal Science. All rights reserved.
INTRODUCTION
J. Anim. Sci. 2007. 85:138–142
doi:10.2527/jas.2006-258
ous 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
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). Previ-
1
Mycobacterium bovis BCG Montreal Strain 9003 was kindly provided by Danuta Radzioch, McGill University, Montreal.
2
We gratefully appreciate the assistance of Doris Hunter with
establishment of the original ear punch fibroblast cell lines; Dana
Dean for electron microscopy and immunocytochemistry; Robert
Schnabel, Jim Derr, and the Core Technologies Lab, Texas A&M
University, with microsatellite analysis; Bhanu Chowdhary for karyotyping; Charles Looney for embryo transfer; and Juan Romano,
Wesley Bisset, Amy Plummer, Peter Rakestraw, and Thomas Kasari
with surgeries and neonatal care of the cloned calf in the Large
Animal Clinic, Texas A&M University.
3
Corresponding author: [email protected]
4
L. G. Adams’ laboratory was supported by the Texas Advanced
Technology Program grant No. 999902-045, USDA, Cooperative State
Research Education and Extension Service grants no. 90-37241-5583
and 93-37204-9491, Texas Agricultural Experiment Station Project
TEXO H-6194 and TEXO H-8409.
Received April 24, 2006.
Accepted August 8, 2006.
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Rescuing valuable genomes by animal cloning
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 ␮g/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 ␮g of estradiol (Sigma), 28 ␮g of
pyruvate (Sigma), and 0.05 ␮g 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 HEPESbuffered TCM 199 supplemented with 10% FCS and
containing (per mL) 5.0 ␮g of cytochalasin B (Sigma)
and 5 ␮g of Hoechst 33342 (Sigma). All oocytes were
carefully selected for the presence of the first polar body
139
and a homogeneous cytoplasm. Enucleation was performed using a beveled 18- to 20-␮m 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-␮m)
size and a morphologically round, smooth shape were
combined with enucleated oocytes. The oocyte-fibroblast 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-␮sec, 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 ␮g 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 ␮M 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 TLHEPES supplemented with 3 mg/mL of BSA. Successfully fused and activated embryos were then placed in
a culture well (Nunc) containing 500 ␮L of TCM 199
supplemented with 10% FCS, 10 ␮g/mL of CHX
(Sigma), and 5 ␮g/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, 5MHz 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 dyelabeled microsatellites were detected using GeneScan
3.1 software (Applied Biosystems). Alleles were as-
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Westhusin et al.
signed 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 intracellular 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 L-glutamine (Gibco), 1 mM
nonessential amino acids (Hazelton Research Products
Inc., Lenexa, KS), 1 mM sodium pyruvate, 7.5% sodium
bicarbonate, and 4% fresh autologous serum.
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 antigenresponsive 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, Cockeysville, 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, L-glutamine,
sodium pyruvate, and sodium bicarbonate (Sigma). Incubation conditions were 37°C with 5% CO2 in air under
humidified conditions.
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 ␮L, 10,000 cells/mL) were transferred to triplicate wells of tissue culture, 60-well, HLA 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 ␮L
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 ␮L 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 ␮L of a 37.5 ␮g/mL solution
of gentamycin sulfate (Gibco) were added to each well
to a final concentration of 25 ␮g/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 ␮L of
0.5% Tween-20 (Sigma) in sterile, distilled water. Well
contents were serially diluted in sterile, distilled water,
and 100 ␮L 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 ␮L 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
141
Rescuing valuable genomes by animal cloning
Table 1. Comparison of the original (Bull 86) with the
cloned (Bull 862) bull for in vitro, peripheral blood-derived, macrophage killing of Brucella abortus, Mycobacterium bovis, and Salmonella dublin1,2
Item
B. abortus
survival, %
S. dublin
survival, %
Clone 862
Replicates
Mean
SD
M. bovis
survival, %
95
52
32
59.6
32.3
50
76
66
64.0
13.1
70
98
52
73.3
23.2
Original 86
Replicates
Figure 1. Cloned Bull 862 at 2 yr of age.
live B. abortus, the data were not normally distributed.
Data were analyzed for significance by the nonparametric Mann-Whitney U-test.
RESULTS
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 pub-
Mean
SD
862 vs. 86
55
67
61
61.0
6.0
P = 0.7
74
59
66
66.3
7.5
P=1
70
92
68
76.7
13.3
P= 1
1
Intracellular survival of each bacterium in peripheral blood monocyte-derived macrophages from Bull 86 and Bull 862 was analyzed
at time 0 h and 12 h postinfection in duplicate culture plates. The
percent survival of each bacterium was determined in triplicate samples (replicates), and the data were analyzed for significance by the
Mann-Whitney U-test.
2
Percent reduction of bacterial survival data for Bull 86 was generated in 1992 before his death, whereas the percent reduction in bacterial survival data for Bull 862 were necessarily determined in 2003
using the identical procedures, instrumentation, and technical personnel.
lished 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).
DISCUSSION
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
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Westhusin et al.
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.
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