Adeno-associated virus-targeted disruption of the CFTR gene in cloned ferrets.
ABSTRACT Somatic cell gene targeting combined with nuclear transfer cloning presents tremendous potential for the creation of new, large-animal models of human diseases. Mouse disease models often fail to reproduce human phenotypes, underscoring the need for the generation and study of alternative disease models. Mice deficient for CFTR have been poor models for cystic fibrosis (CF), lacking many aspects of human CF lung disease. In this study, we describe the production of a CFTR gene-deficient model in the domestic ferret using recombinant adeno-associated virus-mediated gene targeting in fibroblasts, followed by nuclear transfer cloning. As part of this approach, we developed a somatic cell rejuvenation protocol using serial nuclear transfer to produce live CFTR-deficient clones from senescent gene-targeted fibroblasts. We transferred 472 reconstructed embryos into 11 recipient jills and obtained 8 healthy male ferret clones heterozygous for a disruption in exon 10 of the CFTR gene. To our knowledge, this study represents the first description of genetically engineered ferrets and describes an approach that may be of substantial utility in modeling not only CF, but also other genetic diseases.
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ABSTRACT: Ferrets are an important emerging model of cystic fibrosis related diabetes. However, there is little documented experience in the use of advanced techniques to quantify aspects of diabetes pathophysiology in the ferret. Glycemic clamps are the gold standard technique to assess both insulin sensitivity and insulin secretion in humans and animal models of diabetes. We therefore sought to develop techniques for glycemic clamps in ferrets. To assess insulin sensitivity, we performed euglycemic hyperinsulinemic clamps in 5-6 week old ferrets in the anesthetized and conscious states. To assess insulin secretion, we performed hyperglycemic clamps in conscious ferrets. To evaluate responsiveness of ferret islet and entero-insular hormones to low glucose, a portion of the hyperglycemic clamps were followed by a hypoglycemic clamp. The euglycemic hyperinsulinemic clamps demonstrated insulin responsiveness in ferrets similar to that previously observed in humans and rats. The anesthetic isoflurane induced marked insulin resistance, whereas lipid emulsion induced mild insulin resistance. In conscious ferrets, glucose appearance was largely suppressed at 4 mU/kg/min insulin infusion, whereas glucose disposal was progressively increased at 4 and 20 mU/kg/min insulin. Hyperglycemic clamp induced first phase insulin secretion. Hypoglycemia induced a rapid diminishment of insulin, as well as a rise in glucagon and pancreatic polypeptide levels. The incretins GLP-1 and GIP were affected minimally by hyperglycemic and hypoglycemic clamp. These techniques will prove useful in better defining the pathophysiology in ferrets with cystic fibrosis related diabetes.PLoS ONE 01/2014; 9(3):e90519. · 3.53 Impact Factor
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ABSTRACT: Cystic fibrosis is a lethal genetic disorder characterized by viscous mucus and bacterial colonization of the airways. Airway surface liquid represents a first line of pulmonary defense. Studies in humans and animal models of cystic fibrosis indicate that the pH of airway surface liquid is reduced in the absence of cystic fibrosis transmembrane conductance regulator function. Many aspects of the innate host defense system of the airways are pH sensitive, including antimicrobial peptide/protein activity, the rheological properties of secreted mucins, mucociliary clearance, and the activity of proteases. This review will focus on how changes in airway surface liquid pH may contribute to the host defense defect in cystic fibrosis soon after birth. Understanding how changes in pH impact mucosal immunity may lead to new therapies that can modify the airway surface liquid environment, improve airway defenses, and alter the disease course.The international journal of biochemistry & cell biology 01/2014; · 4.89 Impact Factor
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ABSTRACT: Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included hepatic steatosis, hepatic necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (<2 μg elastase-1 per gram of feces). These findings suggest that genetic factors likely influence the extent of exocrine pancreas disease in CF ferrets and have implications for the etiology of pancreatic sufficiency in CF patients. In summary, these studies demonstrate that the CF ferret model develops gastrointestinal pathology similar to CF patients.American Journal Of Pathology 03/2014; · 4.60 Impact Factor
1578? The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 118? ? ? Number 4? ? ? April 2008
Adeno-associated virus–targeted disruption
of the CFTR gene in cloned ferrets
Xingshen Sun,1,2,3 Ziying Yan,1,2 Yaling Yi,1,3 Ziyi Li,1,4 Diana Lei,1 Christopher S. Rogers,5
Juan Chen,1,4 Yulong Zhang,1,2 Michael J. Welsh,2,5,6 Gregory H. Leno,1 and John F. Engelhardt1,2,6
1Department of Anatomy and Cell Biology and 2Center for Gene Therapy, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA.
3College of Life Science, Northeast Agricultural University, Harbin, People’s Republic of China. 4College of Animal Science and Veterinary Medicine,
Jilin University, Changchun, People’s Republic of China. 5Departments of Internal Medicine and Molecular Physiology, Howard Hughes Medical Institute,
Iowa City, Iowa, USA. 6Department of Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.
Cystic fibrosis (CF) is the most common autosomal recessive
condition affecting white individuals. Although the CFTR gene
— which encodes an epithelial chloride channel that is defective in
CF — was cloned nearly 2 decades ago, progress toward treatments
has been hindered by the lack of an animal model that reproduces
the life-threatening lung infections observed in CF patients.
While certain CF mouse models demonstrate phenotypic altera-
tions in response to bacterial agarose bead challenge in the lung
(1), they fail to develop the natural progression of spontaneous
disease seen in humans. This is most likely due to species-specific
expression of alternative chloride channels in their airways (2, 3).
The domestic ferret (Mustela putorius furo) has been considered as
an alternative species for modeling CF, given the high degree of
similarity between ferret and human lung biology and known
utility as a model for other types of lung infection, such as SARS
(4) and influenza (5) virus.
The domestic ferret is an attractive alternative species to model
CF for several reasons. First, ferrets and humans share a remark-
ably similar airway cytoarchitecture (6–8), a feature not shared by
humans and mice. Second, the expression pattern of the CFTR gene
is extremely similar in ferret and human airways (9, 10), with the
highest levels in submucosal glands. Bioelectric and pharmacologic
properties of the CFTR chloride channel in ferret airway epithelia
are also similar to those seen in human airway epithelia (11). In
contrast to those of mice, ferret and human tracheobronchial air-
ways contain abundant submucosal glands that express high levels
of CFTR (9, 10, 12); these glands have been shown to be critical for
airway innate immunity in the ferret (13), as predicted for humans
(14, 15). Gene transfer to ferret airway epithelia with several adeno-
associated virus (AAV) serotypes has also been shown to be very
closely conserved to that seen in human airway epithelia (16); again,
a conservation not shared with mice (3). In addition, the ferret has
a 42-day gestation time and reaches sexual maturity in 5–6 months
(17), making it one of the more rapidly reproducing species for ani-
mal modeling by somatic cell nuclear transfer (SCNT). Together,
these studies point to the potential of the ferret to be a good species
on which to model CF lung disease in humans.
Since the birth of Dolly (18), the concept of combining SCNT
with gene targeting in somatic cells has held tremendous poten-
tial for the development of new animal models of human disease
such as CF. However, to date, this approach has failed to deliver
on such potential due to technical challenges. A few laboratories
have successfully applied SCNT with gene-targeting technology in
livestock for agricultural and biomedical (organ transplantation
and protein production) applications (19–22). However, to our
knowledge, no report has yet to apply this application to gener-
ate better non-rodent disease models. The development of robust
gene-targeting technologies that are compatible with SCNT have
also been rate limiting, slowing progress in this area. Additionally,
efficient nuclear transfer (NT) cloning procedures for potentially
useful species such as ferret have lagged behind those for larger
species such as sheep (8%–10%) (18, 23), cattle (10%–20%) (24,
25), and pigs (5.5%) (26). These cloning efficiencies of 5%–20%
have been sufficient to generate gene-targeted sheep, cattle, and
pigs (19–22). While ferret cloning from highly reprogrammable
somatic cumulus cells has been reported (27), SCNT methods
with fibroblasts (the best cell type for gene targeting) have yet
to be developed. Thus, further optimization of fibroblast-based
SCNT cloning procedures are needed to facilitate the develop-
ment of genetic models in the ferret.
Nonstandard?abbreviations?used: AAV, adeno-associated virus; CF, cystic fibrosis;
NT, nuclear transfer; rAAV, recombinant AAV; SCNT, somatic cell nuclear transfer.
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article:?J. Clin. Invest.?118:1578–1583 (2008). doi:10.1172/JCI34599.
? The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 118? ? ? Number 4? ? ? April 2008
Here we report the production of cloned domestic ferrets hetero-
zygous for a disrupted CFTR gene. Critical to this success was the
development of recombinant AAV (rAAV) gene-targeting methods
in fetal ferret fibroblasts and a serial NT cloning technique to reju-
venate senescent gene-targeted cells prior to cloning of full-term fer-
rets. Using this serial cloning technique, 8 healthy CFTR gene–dis-
rupted male ferrets were obtained from 11 recipient jills. This study
has demonstrated the feasibility of AAV-mediated genetic manipula-
tion in the ferret using SCNT. Such an approach may also be of util-
ity in genetic manipulation and disease modeling in other species.
rAAV-mediated targeting of the CFTR gene in ferret fetal fibroblasts. rAAV
has been previously shown to facilitate homologous recombina-
tion between its viral DNA and cellular genomic DNA of infected
fibroblasts (28). Therefore, we chose to use this virus to target the
CFTR gene in ferret fibroblasts. A ferret BAC library (CHORI-237)
was constructed by the BACPAC Resources Center, Children’s Hos-
pital Oakland Research Institute, and used to isolate an approxi-
mately 150-kb genomic fragment containing exon 10 of the CFTR
gene. Genomic sequences from this BAC clone were used to gener-
ate a rAAV targeting vector harboring a PGK promoter–driven neo-
mycin resistance gene cassette flanked by sequences encompass-
ing CFTR exon 10 and the adjacent introns (Figure 1). Male fetal
fibroblasts derived from 28-day fetuses were used for CFTR gene
targeting with rAAV serotype-2 (rAAV2). Fibroblasts were infected
with rAAV2 virus at a multiplicity of infection of 100,000 particles
per cell and subsequently serially diluted into 96-well plates and
placed under G418 selection. During optimization of this proce-
dure, we found that the number of cells seeded into each well and
the timing of G418 section after plating were the most critical vari-
ables to the subcloning process, which targeted 15%–30% of wells
with surviving clones by 15 days. Typically, seeding 200–500 cells
per well and initiation of selection (300 μg/ml G418) at day 2 fol-
lowing passaging into 96-well plates was optimal.
Following replica plating of each primary 96-well plate of selected
fibroblast clones, a single plate was used for PCR screening of
flanking sequences outside each targeting arm of the vector (Fig-
ure 1A). In total, approximately 500 clones were typically screened
in a single experiment. Although the efficiency of gene targeting
ranged from 0.5%–2% depending on the experiment, we found that
the largest hurdle was the rapid senescence of PCR-positive tar-
geted clones once they were expanded to 24-well plates. Initially,
this was an obstacle that prevented direct confirmation of gene
targeting by Southern blotting prior to SCNT.
Rejuvenation of PCR-positive CFTR-targeted senescent fibroblasts by
SCNT. CFTR-targeted ferret fibroblast clones senesce rapidly dur-
ing expansion following the initial round of PCR screening; there-
fore, it was necessary to develop methods for rejuvenating and
expanding these candidate CFTR-targeted clones for Southern
blot confirmation of the CFTR-targeting events. To this end, we
performed several rounds of SCNT on 3 independent PCR-posi-
tive CFTR-targeted clones and successfully cloned six 21-day ferret
fetuses by NT (Table 1). Each of these NT fetuses was dissociated
with trypsin, and primary fetal fibroblasts were generated under
selection with G418. DNA was then derived from each of these pri-
mary fibroblast lines and used for Southern blotting. From these
cultures, one of the 3 original senescent fibroblast lines (CL-B96)
gave rise to secondary NT-derived fetal fibroblasts with a “clean”
CFTR gene-targeting event and no other integrations, as shown by
Southern blotting (Figure 1B). The remaining 2 senescent fibro-
blast lines gave rise to NT-derived fetal fibroblasts that were neo-
mycin resistant but had an insertion of the rAAV vector at a non-
homologous site in the genome. NT-derived fetal fibroblasts from
the CL-B96 rejuvenated fibroblast clone were expanded to passage
5 for SCNT cloning of live-birth ferrets.
SCNT cloning of CFTR-targeted ferrets from fetal fibroblasts. We have
reported methods of SCNT cloning of ferrets using cumulus cells
(27). However, in initial pilot studies, we observed that these pre-
vious methods were not compatible with efficient cloning from
fetal fibroblasts. Thus, ferret SCNT protocols were optimized to
resolve the major deficiencies preventing successful ferret cloning
with fetal fibroblasts. Major variables included the age of recipi-
ent oocytes, alterations to media designed to reduce the stress
CFTR gene targeting in ferret fibroblasts. (A) Genomic fragment containing exon 10 of
the ferret CFTR gene (top) and the rAAV CFTR targeting vector (bottom). Arrows mark
nested primers used for PCR screening of targeting events. Restriction sites and probes
used for Southern blot confirmation of targeting events are also shown. (B) Southern
blot analysis of fibroblasts derived from the CL-B96 CFTR gene–targeted 21-day NT
cloned ferret embryo using restriction sites and probes marked in A. The originating
fibroblast line used for targeting is shown in lanes 1 and 4, while the CFTR gene targeted
fibroblasts are shown in lanes 2 and 3. Arrowheads to the right of blots indicate the non-
targeted (filled) and gene-targeted (open) CFTR alleles.
1580?The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 118? ? ? Number 4? ? ? April 2008
during embryo manipulation, and optimization of the timing for
oocyte activation to more efficiently promote nuclear remodeling.
Using this optimized method for ferret fibroblasts, we observed
a doubling in oocyte implantation as compared with the previ-
ous methods developed for cumulus cells (Table 2). This enhanced
level of implantation also led to normal fetal development in 2.2%
of implanted reconstructed embryos, whereas fetal development
failed to occur when we used the previous methods employing
cumulus cells (Table 2).
Using this improved method of SCNT with ferret fibroblasts,
we proceeded to clone live-birth ferrets from the NT-rejuvenated,
CFTR-targeted fibroblasts (CL-B96). In total, 11 recipient jills
were each adoptively transferred with 35–60 reconstructed NT
embryos derived from the CL-B96 line and allowed to develop to
term (Table 3). Fourteen live pups were born by natural birth or
C-section from these 11 jills. Twelve of these 14 pups appeared
to be healthy at birth. In 3 cases however, the jills only nursed
one pup in the litter or failed to nurse at all. Two abandoned
pups were rescued by transfer to foster jills. However, it was not
possible to do this in all cases of parental neglect, since surrogate
jills were not always available. Two of the 14 pups born appeared
weak and died; however, it was unclear whether the jills were lac-
tating and surrogates were not available at the time. Southern
blotting confirmed that all 8 of the surviving NT cloned ferrets
(Figure 2A) were heterozygous for a single targeted allele of the
CFTR gene (Figure 2B). Furthermore, these ferrets all remained
healthy and had preweaning growth rates similar to those of
noncloned pups (Figure 2C).
Combining rAAV-mediated gene targeting with SCNT cloning lays
the foundation for numerous other applications in disease mod-
eling with ferrets and other species (see the companion article in
this issue; ref. 29). In previous reports of the generation of gene-
targeted animals in pig (20, 21), cow (22), and sheep (19), linear
fragments have been used to facilitate gene targeting with non-
viral gene transfer methods. Important differences between these
previous approaches and our current strategy are worth noting.
First, gene-targeting experiments in pig and sheep fibroblasts have
utilized expression from the endogenous promoter of the target
gene to facilitate positive selection by insertion of an internal ribo-
some entry site upstream to the resistance marker gene (19–21).
This was not feasible for targeting the CFTR locus, since this gene
is not expressed in fibroblasts. A second strategy used to target
the bovine gene encoding immunoglobulin-μ required an alterna-
tive approach, since this gene is not expressed in fibroblasts (22).
In this context, a diphtheria toxin A gene was used as a negative
marker to select against random integration events and led to a
targeting efficiency of approximately 0.5%. This efficiency of tar-
geting appears to be similar to that obtained in our experiments to
target the ferret CFTR allele using rAAV.
Selection of gene-targeted fibroblasts can lead to rapid senes-
cence. This was indeed the case in the ferret, where CFTR-targeted
fibroblast clones rarely expanded beyond 1 × 106 cells. Factors
affecting senescence appear to be linked to neomycin selection
and stress induced by this process, as serial dilution cloning of
nonselected fibroblast could easily be expanded to greater than
5 × 107 cells. This phenomenon may be one reason why there are
few reports of gene-targeted animals produced by SCNT. Reversal
of this phenotype in fibroblasts by NT, as described in this report,
indicates that the causes of selection-based senescence are not per-
manent. Cell rejuvenation provides an alternative strategy to more
efficiently produce gene-targeted animal models. Interestingly,
senescence of CFTR-targeted fibroblasts was not a major obstacle
in the cloning of CF pig models (see the companion article in this
issue; ref. 29), suggesting that species-specific factors likely influ-
ence the biology of selection-induced senescence.
The creation of both ferret and pig CF models provides new
opportunities for dissecting the pathophysiology of CF and test-
ing of new therapies not previously approachable in mouse mod-
els of this disease. Furthermore, the fact that AAV-mediated gene
targeting was successfully used to genetically engineer both fer-
ret and pig models suggests that this technology may be gener-
ally applicable to modeling in any species. It remains unknown
Rejuvenation of senescent CFTR gene–targeted ferret fibroblasts by SCNT
No. of NT
No. of recipients
No. of 21-day
No. of Neo-resistant
Southern blot positive
for CFTR targeting
Optimization of SCNT procedures with ferret fibroblasts
No. of oocytes
No. of oocytes
No. of embryos
No. of recipients
No. of No. of
AMethods used for cumulus cell–based SCNT as previously reported (27). BMethods developed in the current report for fibroblast-based SCNT.
? The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 118? ? ? Number 4? ? ? April 2008
whether homozygous CFTR gene–disrupted ferrets will contract
lung disease identical to that of humans. However, regardless of
their phenotype, it is likely the field will learn much about CFTR
biology from this model, as has been the case for CF mice. Under
the proper light cycle, ferrets can reach sexual maturity in approxi-
mately 5–6 months. Hence, with a gestation time of 42 days, CFTR-
deficient ferrets could be available in approximately 1–1.5 years.
Since ferrets are a preferred model for other devastating infectious
human lung diseases, such as H5N1 influenza (5) and SARS virus
(4), the ability to generate genetically engineered ferrets may also
be of significant utility to pandemic viral disease research.
Animals. Ferrets were purchased from Marshall Farms. Female sable jills
(virgin, 6–7 months of age) and albino jills (primipara, 9–12 months of
age) were in estrus when delivered. Vasectomized male ferrets (albino, 12
months of age) were used for mating to induce follicular oocyte matura-
tion in oocyte donor jills and to induce pseudopregnancy in NT embryo
surrogate jill recipients. All ferrets were housed in separate cages under
controlled temperature (20–22°C) and long daylight cycle (16 hours light/
8 hours dark). Ferret chow was obtained from Marshall Farms. The use of
animals in this study was carried out according to a protocol approved by
the University of Iowa Institutional Animal Care and Use Committee and
conformed to or exceeded NIH standards.
Collection of fetal ferret fibroblast. Fetal ferret fibroblasts were obtained
from 28-dpc fetuses derived from a sable (female) × sable (male) mating
(Marshall Farms) as previously described (30). Each fetus was treated indi-
vidually. Karyotype analysis was performed on each embryo line, and only
male fibroblasts with a normal chromosomal profile were used for SCNT
and CFTR gene targeting with rAAV.
Cloning ferret CFTR genomic DNA. Ferret genomic DNA was extracted from
internal organs collected from an E28 female fetus and used to construct a
ferret genomic BAC library through the BACPAC Resources Center (http://
bacpac.chori.org) at Children’s Hospital Oakland Research Institute. The
average length of the ferret genomic DNA inserts in this library is approxi-
mately 150 kbp. BAC clones encompassing ferret CFTR exon 10 were iso-
lated from this library after screening with the CFTR exon 10 probe, synthe-
sized according to the partial ferret CFTR cDNA sequence (gb:S82688) (9).
BAC DNA from the CFTR-positive clone was prepared with the ΨCLONE
BAC DNA isolation kit (Princeton Separations). Sequencing of the CFTR
exon 10 and adjacent introns was initiated with 2 primers located inside
the CFTR exon 10 region (primers: e10F: 5′-TGATGATTATGGGAGAGTT-
GGAGCC-3′ and e10R: 5′-GCATGCTTTGATGACACTCCTG-3′). Primer
walking was used to sequence approximately 2 kb on each side of exon
10 to generate a contig for cloning of the targeting vector. Based on the
obtained CFTR genomic sequence, primers for subcloning were designed,
and two 2.0-kb CFTR fragments containing exon 10 and flanking intronic
sequence were retrieved from BAC DNA by PCR with AccuPrime Pfx Super-
Mix (Invitrogen). The PCR products were cloned into the pBlunt4PCR
vector with Topo cloning kit (Invitrogen) and confirmed by sequencing.
The resultant plasmids were designated as pTopo-Left and pTopo-Right,
encompassing exon 10 and left-arm or right-arm introns, respectively.
Generation of CFTR-targeting proviral vector. To construct the AAV target-
ing vector centered on CFTR exon 10, a 0.97-kb left homologous arm and
a 1.23-kb right homologous arm were retrieved by PCR from pTopo-Left
and pTopo-Right, respectively. The primer set for the left arm was: 5′-ccatc-
gatGGCACCCCTGTGTTATCTTTCT-3′ (forward) and 5′-ccggtacctatca-
GATCCAGGAAAACTGAGAGCAG-3′ (reverse). The right-arm set was:
and 5′-ggactagtggatccGATGGCCTTTCCTTTGGATGGA-3′ (reverse) (low-
ercase letters indicate restriction enzyme sequences introduced for clon-
ing). The reverse primer of the left arm and forward primer of the right
arm are located at the center of exon 10. The 2 PCR products together with
a 1.7-kb PGK promoter–driven neomycin resistance expression cassette
were assembled and finally cloned into an AAV2 proviral plasmid, giving
rise to vector harboring 2.3-kb ferret CFTR genomic DNA with a neomy-
cin cassette inserted at the center of exon 10. The rAAV2 targeting virus
(AV.CFtarg) was produced as previously described using a triple plasmid
transfection procedure in 293 cells and purified over an iodixanol cushion
followed by ion exchange HPLC (31).
Screening for CFTR-targeted fibroblast clones. Targeting was initiated by
infecting ferret primary fibroblasts derived from a male E28 fetus with
AV.CFtarg at a multiplicity of infection of 100,000 particles per cell. On
day 1 following infection, fibroblasts were subsequently serially diluted
into twenty 96-well plates at 200–500 cells per well. These seeding densi-
ties allowed approximately 15%–30% of wells to give rise to G418-resistent
clones. Selection was initiated on day 2 following replating by the addition
of 300 μg/ml G418 to the media, and cells were cultured for an additional
15 days. Typically, this screening gave rise to approximately 500 G148-
resistant clones, which were subsequently expanded into 3 replica 96-well
plates. Once these replica plates reached confluence, a single plate was used
for PCR screening of flanking genomic sequences outside each targeting
arm of the vector and anchored sequences within the vector. Nested PCR
screening was then performed for the predicted left-side homologous
recombination event. Cells in the 96-well plates were directly lysed with 10
μl per well of lysis buffer (50 mM KCl, 1.5 mM MgCl2, 10 mM Tris, pH 8.5,
0.5% NP40, 0.5% Tween-20), and 10% of the cell lysate (1 μl) was used for
PCR with the first-round PCR primer set: F1 (5′-TGGTTTCAAGGGAAT-
GGGGTC-3′) and intR1 (5′-AAGCGAAGGAGCAAAGCTGCTA-3′). Two
percent of the first-round PCR products was then used as template for
the second-round PCR with primers: F2 (5′-GGTGCAGGAGGTGTTTT-
GTCATAGA-3′) and intR2 (5′-GCTAAAGCGCATGCTCCAGACT-3′). The
positive clones were further confirmed by another nested PCR reaction
against the right arm of the integration site using the first-round primer
set: intF1 (5′-CGGACCGCTATCAGGACATAG-3′) and R1 (5′-TACGAAAT-
GCAGCAAGCGCC-3′); and the second-round nested primer set: intF2
(5′-AGGTGTCATTCTATTCTGGGG-3′) and R2 (5′-CCCAGGCATCCCT-
GAAACT-3′). The clones that were PCR positive for both the left and right
Cloning of CFTR-targeted ferrets by SCNT
Total (n = 11)
3.0 ± 0.6
APercent efficiency of transferred oocytes that gave rise to live births.
BOne abandoned pup was transferred to a foster jill. CDespite the fact all
pups were born healthy, the jill only nursed one pup and neglected oth-
ers or failed to nurse all pups in the litter.
1582? The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 118? ? ? Number 4? ? ? April 2008
arms of the predicted integration event were expanded to 24-well plates
prior to NT rederival of 21-day embryo fibroblasts. Fibroblasts derived
from cloned 21-day embryos were used for isolation of genomic DNA and
Southern blot confirmation of the integration event. Genomic DNAs were
digested with either AflII (which has a unique site in the targeting vector)
or BamHI (which does not cut within the targeting vector). Southern blot-
ting was visualized with a 32P-labeled CFTR probe against the 5′ upstream
intronic CFTR sequence of the left homologous arm or a Neo probe against
the neomycin cDNA.
Rejuvenating fibroblast lines using SCNT. Rederival of highly proliferative
fetal fibroblasts from PCR-positive CFTR gene–targeted senescent cells was
accomplished using SCNT. Immature oocytes were obtained from sable
jills mated with vasectomized male ferrets 24 hours prior to collection.
To retrieve the oocytes, jills were euthanized by administration of pento-
barbital sodium injection (50–100 mg/kg, i.p). Preovulated follicles were
punctured with fine forceps in mPBS (Dulbecco PBS supplemented with
0.1% [wt/vol] d-glucose, 36 mg/l pyruvate, and 0.4% [wt/vol] BSA) to release
the cumulus-oocyte complexes (COCs). COCs were cultured in TCM-
199 plus 10% FBS plus 10 IU/ml equine chorionic gonadotrophin (eCG;
G4527; Sigma-Aldrich) plus 5 IU/ml of human
chorionic gonadotrophin (hCG; C8554; Sigma-
Aldrich). For enucleation, oocytes were trans-
ferred to mPBS medium containing 7.5 μg/ml
cytochalasin B (CB; C6762; Sigma-Aldrich) in
the micromanipulation chamber. Using Nomar-
ski optics, the first polar body and chromosome
spindle were aspirated with a minimal volume
of oocyte cytoplasm with a 20-μm (inside diam-
eter [ID]) PeizoDrill glass pipette (Humagen).
A senescent fibroblast (diameter ≥40 μm) was
inserted into the perivitelline space (PVS) of
enucleated oocytes using another pipette (35
μm ID). This larger-diameter pipette was spe-
cifically needed to accommodate the larger size
of senescent fibroblasts. The NT-reconstructed
embryos were transferred into fusion medium
(0.3–0.26 M mannitol, 0.1 mM MgCl2, 0.1 mM
CaCl2, 0.5 mM HEPES, 0.01% [wt/vol] BSA),
placed between 2 parallel electrodes and subject-
ed to an electrical pulse of 1 DC of 180 V/mm
for 30 μs from an ECM 2001 (BTX). The fused
embryos were then activated with 5 mM iono-
mycin for 4 minutes, then 2 mM 6-dimethylaminopurine for 3 hours. Acti-
vated embryos were cultured for 24 hours and then were transferred into
pseudopregnant albino recipient jills. A pseudopregnant state was achieved
in surrogate albino virgin jills through mating with a vasectomized albino
male 24 hours prior to embryo transfer. At E21 the fetuses were collected
and fetal fibroblasts were established as described above, except 300 μg/ml
G418 was added to the media. Once expanded, these cells were then used for
Southern blot screening of the CFTR gene targeting event.
Cloning of CFTR-targeted ferrets by SCNT. The procedure described for reju-
venating fibroblast lines by SCNT was also used for cloning live-birth fer-
rets from Southern blot–confirmed E21 CFTR gene–targeted fibroblasts
(CL-B96 clone) with minimal modification. Briefly, the same pipette was
used for both enucleating the oocyte and insertion of fibroblasts into the
PVS of enucleated oocytes. All other NT procedures were identical to those
described above. The reconstructed embryos were transferred into albino
primipara pseudopregnant jills. If natural birth did not occur at 42 days
gestation, the recipients were treated with prostaglandin (Lutalyse; Pfizer;
0.5 mg to 1 mg i.m.) to induce labor. If no kits were delivered within
3 hours, 0.3 ml of oxytocin was subsequently administered to the jill.
Cloning of CFTR-targeted ferrets. (A) Cloned
CFTR-targeted ferrets. The top left panel
shows the first clone (sable coat color) at 5
weeks of age with its albino noncloned foster
sibling. The other panels show sable clones
with ages indicated. (B) Southern blot analy-
sis of ear fibroblast DNA from the 8 CFTR-
targeted cloned ferrets (nos. 1–8) using
AflII-digested genomic DNA and the indi-
cated CFTR and neomycin probes. NT, non-
targeted unrelated ferret DNA. Arrowheads
indicate the nontargeted (filled) and gene-tar-
geted (open) CFTR alleles. (C) Preweaning
growth rate of CFTR-targeted ferret clones
as compared with unrelated noncloned fer-
rets. Results are shown as the mean ± SEM
for the indicated n in each group.
? The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 118? ? ? Number 4? ? ? April 2008
If these treatments were unsuccessful to induce labor within 8 hours, a
C-section was performed. If the recipients failed to produce enough milk to
feed kits, the young were fostered onto another jill when available.
The Cystic Fibrosis Foundation (J.F. Engelhardt and G.H. Leno);
NIH grants HL61234 (to M.J. Welsh) and DK047967 (to J.F. Engel-
hardt); and the Center for Gene Therapy (DK054759) funded this
research. The authors want to thank R. Scipioni Ball and her staff at
Marshall Farms for their kind assistance with ferret care and repro-
duction. The authors also thank the personnel in the University of
Iowa Animal Facility for their efforts in maintenance of the ferret
colony and Shiva Patil and Heather Major for fibroblast karyotyp-
ing through the University of Iowa Children’s Hospital Cytogenet-
ics Laboratory. We also gratefully acknowledge the assistance of
Pieter J. de Jong (director) and Jeff Garnes at the BACPAC Resources
Center at Children’s Hospital Oakland Research Institute for gen-
erating the ferret BAC library funded through an NHGRI-NIH
White Paper Proposal (G.H. Leno and J.F. Engelhardt).
Received for publication November 28, 2007, and accepted in
revised form January 23, 2008.
Address correspondence to: John F. Engelhardt, Room 1-111 BSB,
Department of Anatomy and Cell Biology, University of Iowa
Carver College of Medicine, 51 Newton Road , Iowa City, Iowa
52242, USA. Phone: (319) 335-7744; Fax: (319) 335-6581; E-mail:
Xingshen Sun and Ziying Yan contributed equally to this work.
1. Heeckeren, A., et al. 1997. Excessive inflammatory
response of cystic fibrosis mice to bronchopul-
monary infection with Pseudomonas aeruginosa.
J. Clin. Invest. 100:2810–2815.
2. Grubb, B.R., Paradiso, A.M., and Boucher, R.C.
1994. Anomalies in ion transport in CF mouse tra-
cheal epithelium. Am. J. Physiol. 267:C293–C300.
3. Liu, X., Yan, Z., Luo, M., and Engelhardt, J.F. 2006.
Species-specific differences in mouse and human
airway epithelial biology of recombinant adeno-
associated virus transduction. Am. J. Respir. Cell Mol.
4. Martina, B.E., et al. 2003. Virology: SARS virus
infection of cats and ferrets. Nature. 425:915.
5. Subbarao, K., and Luke, C. 2007. H5N1 viruses and
vaccines. PLoS Pathog. 3:e40.
6. Mercer, R.R., Russell, M.L., Roggli, V.L., and
Crapo, J.D. 1994. Cell number and distribution in
human and rat airways. Am. J. Respir. Cell Mol. Biol.
7. Leigh, M.W., et al. 1986. Postnatal development of
tracheal surface epithelium and submucosal glands
in the ferret. Exp. Lung Res. 10:153–169.
8. Wang, X., Zhang, Y., Amberson, A., and Engelhardt,
J.F. 2001. New models of the tracheal airway define
the glandular contribution to airway surface fluid
and electrolyte composition. Am. J. Respir. Cell Mol.
9. Sehgal, A., Presente, A., and Engelhardt, J.F. 1996.
Developmental expression patterns of CFTR in fer-
ret tracheal surface airway and submucosal gland
epithelia. Am. J. Respir. Cell Mol. Biol. 15:122–131.
10. Engelhardt, J.F., et al. 1992. Submucosal glands are
the predominant site of CFTR expression in the
human bronchus. Nat. Genet. 2:240–248.
11. Liu, X., Luo, M., Zhang, L., Ding, W., Yan, Z., and
Engelhardt, J.F. 2007. Bioelectric properties of chlo-
ride channels in human, pig, ferret, and mouse air-
way epithelia. Am. J. Respir. Cell Mol. Biol. 36:313–323.
12. Choi, H.K., Finkbeiner, W.E., and Widdicombe, J.H.
2000. A comparative study of mammalian tracheal
mucous glands. J. Anat. 197:361–372.
13. Dajani, R., et al. 2005. Lysozyme secretion by sub-
mucosal glands protects the airway from bacterial
infection. Am. J. Respir. Cell Mol. Biol. 32:548–552.
14. Verkman, A.S., Song, Y., and Thiagarajah, J.R. 2003.
Role of airway surface liquid and submucosal
glands in cystic fibrosis lung disease. Am. J. Physiol.
Cell Physiol. 284:C2–C15.
15. Wine, J.J., and Joo, N.S. 2004. Submucosal glands
and airway defense. Proc. Am. Thorac. Soc. 1:47–53.
16. Liu, X., et al. 2007. Comparative biology of rAAV
transduction in ferret, pig and human airway epi-
thelia. Gene Ther. 14:1543–1548.
17. Fox, J.G., and Bell, J.A. 1998. Growth, reproduction,
and breeding. In Biology and diseases of the ferret. J.G.
Fox, editor. Williams & Wilkins. Baltimore, Mary-
land, USA. 211–227.
18. Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J.,
and Campbell, K.H. 1997. Viable offspring derived
from fetal and adult mammalian cells. Nature.
19. McCreath, K.J., et al. 2000. Production of gene-
targeted sheep by nuclear transfer from cultured
somatic cells. Nature. 405:1066–1069.
20. Lai, L., et al. 2002. Production of alpha-1,3-galac-
tosyltransferase knockout pigs by nuclear transfer
cloning. Science. 295:1089–1092.
21. Dai, Y., et al. 2002. Targeted disruption of the
alpha1,3-galactosyltransferase gene in cloned pigs.
Nat. Biotechnol. 20:251–255.
22. Kuroiwa, Y., et al. 2004. Sequential targeting of the
genes encoding immunoglobulin-mu and prion
protein in cattle. Nat. Genet. 36:775–780.
23. Schnieke, A.E., et al. 1997. Human factor IX trans-
genic sheep produced by transfer of nuclei from
transfected fetal fibroblasts. Science. 278:2130–2133.
24. Wells, D.N., et al. 2003. Coordination between
donor cell type and cell cycle stage improves
nuclear cloning efficiency in cattle. Theriogenology.
25. Yang, X., et al. 2007. Nuclear reprogramming of
cloned embryos and its implications for therapeu-
tic cloning. Nat. Genet. 39:295–302.
26. Walker, S.C., et al. 2002. A highly efficient method
for porcine cloning by nuclear transfer using in vitro-
matured oocytes. Cloning Stem Cells. 4:105–112.
27. Li, Z., et al. 2006. Cloned ferrets produced by somat-
ic cell nuclear transfer. Dev. Biol. 293:439–448.
28. Russell, D.W., and Hirata, R.K. 1998. Human gene
targeting by viral vectors. Nat. Genet. 18:325–330.
29. Rogers, C.S., et al. 2008. Production of CFTR-null
and CFTR-ΔF508 heterozygous pigs by adeno-asso-
ciated virus–mediated gene targeting and somatic
cell nuclear transfer. J. Clin. Invest. 118:1571–1577.
30. Li, Z., et al. 2005. Nuclear transfer of M-phase fer-
ret fibroblasts synchronized with the microtubule
inhibitor demecolcine. J. Exp. Zoolog. A Comp. Exp.
31. Yan, Z., et al. 2006. Unique biologic properties of
recombinant AAV1 transduction in polarized human
airway epithelia. J. Biol. Chem. 281:29684–29692.