Theriogenology 67 (2007) 127 133
www.theriojournal.com
Transgenic animal production and animal biotechnology
*
J.M. Robl , Z. Wang, P. Kasinathan, Y. Kuroiwa
Hematech Inc., 4401 South Technology Dr., Sioux Falls, SD 57106, USA
Abstract
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.
# 2006 Elsevier Inc. All rights reserved.
Keywords: Gene transfer; Viral vectors; Sperm-mediated gene transfer; Gene targeting; Artificial chromosome; Animal biotechnology
1. Introduction goats [3], sheep [4] and pigs [5] for commercial
production of highly valuable human therapeutics.
Production of transgenic livestock was demonstrated Substantial improvements have been made in trans-
to be feasible over two decades ago [1]. It became genic technology over the past 20 years. Several DNA
apparent almost immediately that the method used to transfer systems are now available that greatly improve
produce the transgenic livestock had substantial efficiency, thereby reducing the cost associated with
limitations that would impede its use both for research transgenic animal production. Methods are now available
and commercial applications. The method, pronuclear for targeting foreign DNA to specific sites in the genome
microinjection, was highly inefficient and, therefore, [6 10], alleviating concerns about harmful mutations and
costly. Efficiencies of<1% required microinjection and allowing the inactivation of endogenous genes. Size of
transfer of thousands of embryos to produce a few foreign DNA is also no longer a limitation because it is
transgenic offspring. A second limitation of the now possible to insert stable microchromosome vectors
pronuclear microinjection method was the random carrying Mbs of DNA [11]. Three alternative methods for
integration of a highly variable number of copies of the DNA transfer, i.e. transfer using viral vectors, sperm-
transgene, which resulted in variable expression. The mediated DNA transfer and somatic cell cloning, are
requirement for integration placed some constraints on discussed in the following sections. This review focuses
the length of the sequence that could be transferred and on the large mammalian species of livestock, but
random integration could result in harmful mutations. acknowledges that substantial progress has also been
Despite these drawbacks, pronuclear microinjection made in transgenic technology for poultry and fish.
was successfully used to generate transgenic cattle [2],
2. DNA transfer using viral vectors
* Corresponding author. Tel.: [phone]x1511;
Interestingly, the first successful foreign DNA
fax: [phone].
E-mail address: [e-mail] (J.M. Robl). transfer in a mammal was accomplished using viral
0093-691X/$  see front matter # 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2006.09.034
128 J.M. Robl et al. / Theriogenology 67 (2007) 127 133
DNA. In 1974, Jaenisch and Mintz showed that adenoviruses (DNA viruses) were also used as vectors
seemingly healthy mice, carrying copies of foreign to make transgenic mice [21], rats and cattle [22].
viral DNA, could be produced by microinjecting SV40 Although transgenic mice, with a propensity for
DNA into the blastocysts [12]. Subsequently, it was germline transmission were produced [21], similar
shown that germline transmission of viral DNA into the success has not been shown in other species [22].
animal genome could be achieved by directly infecting The real breakthrough in using viral vectors to make
preimplantation stage embryos with Moloney leukemia transgenic animals came from the development of
viruses (MLV) [13]. Such pioneering work led to lentiviral vectors [23,24] and their subsequent use in
numerous successes of making transgenic animals using making transgenic animals [16,17]. Because of their
a retroviral genome as vector to deliver transgenes. capacity for infecting a large spectrum of host cells and
So far, two types of retroviral vectors have been cells at different cycle stages, lentiviral transgenesis is
developed for making transgenic animals; vectors currently the most efficient method for making
derived from the genome of prototypic retrovirus (such transgenic animals, including livestock and poultry.
as MLV) and vectors derived from the genome of a more Typically, a lentiviral vector system contains separate
complex retroviruses (such as lentiviruses). Compared vector and packaging constructs, with the vector
to the prototype retroviruses, lentiviruses contain a carrying the transgene of interest and the packaging
more complex genome, carrying at least three addi- plasmids carrying the viral proteins required for
tional genes [14]. However, the most substantial packaging. To increase the expression of transgenes,
difference between prototypic retroviruses and lenti- lentiviral vectors have been developed which incorpo-
viruses in using them as vectors to deliver transgenes is rate enhancer elements. To reduce the effect of
that the lentiviral genome can be actively transported misregulating host genes by the viral LTRs, viral
into the nucleus, allowing it to deliver transgenes to enhancer and promoter sequences within the LTRs have
non-dividing cell types [15]. been deleted and subsequently replaced by either
Before the first reports of using lentiviral vectors to ubiquitous promoter sequences or by tissue-specific
make transgenic animals [16,17], retroviral vectors promoter sequences [17,25,26]. In some cases, lenti-
based on prototypic retroviruses such as MLV were the viral vectors carrying tetracycline (tet) regulatable
main retroviral vectors used in transgenic research. transgenes were also developed so that the expression of
Although introducing transgenes into the host genome the transgene could be regulated at will [27].
by retroviral vectors was successful in many animal The list of livestock species from which transgenic
species, including livestock, the method has some animals were produced by a lentiviral vector-based
serious limitations. First, the transgenes delivered by a approach is growing. It was reported recently that using
retroviral vector cannot be expressed in the transgenic lentiviral vectors, transgenic pigs could be produced
animal. Silencing of the transgene has been shown to be with high efficiency; 13 [28] and 31% [29]. Further-
caused by the recruitment of gene repression machinery more, it was shown that more than 90% of the
in the host cells by the promoter and enhancer transgenic animals expressed the transgene at a high
sequences of the retroviral long-terminal-repeats level. This approach was also successful in cattle. When
(LTRs) and the subsequent hypermethylation of the lentiviral vectors carrying transgenes were used for
viral promoter sequences by de novo DNA methylation infecting oocytes before fertilization, all of the resulting
[18]. Second, due to the small size in volume of such calves from the transgenic positive embryos expressed
prototypic viruses, it is difficult to package a transgene the transgene [30]. It is interesting to note, however, that
larger than 10 kb, which seriously limits its application when preimplantation stage bovine embryos were
for delivering large genes [19]. Third, it was shown that infected with the same lentiviral vector, no transgenic
the LTRs can interfere with mammalian promoters to calves were produced [30].
suppress or misdirect host gene expression [20]. Fourth, Despite the advantages in using lentiviral vectors,
since infection by prototype retroviruses requires the this approach has limitations. Due to the size of the
breakdown of the nuclear envelope during mitosis, RNA genome, lentiviral vectors still have the limitation
infection of early embryos by such viruses often show a of about 10 kb of transgene DNA, similar to conven-
delay in transgene integration, therefore producing tional prototypic retroviral vectors. The success of using
mosaic animals in which germline transmission of the lentiviral vectors in certain species seems not to
transgene was very low. guarantee success in other species. Despite its success
To overcome the limitation set by retroviruses in in mouse, rat, pig, cattle and chicken [16,17,30,31], no
infecting non-dividing cells, replication defective transgenic monkeys were made by lentiviral transfer
J.M. Robl et al. / Theriogenology 67 (2007) 127 133 129
[32]. Another limitation, which is common to all surface antigen on sperm before being incubated with
transgenic methods that involve random insertion into sperm for fertilization. When sperm, treated in this way,
an endogenous chromosome, is its potential for were used to fertilize mouse and pig eggs, transgenic
mutating an endogenous genome. offspring were produced with high efficiency [44].
Modest success has also been achieved in sperm-
3. Sperm-mediated DNA transfer mediated gene transfer by direct injection of foreign
DNA into the reproductive tract of males before mating
Brackett et al. [33] first demonstrated that mamma- [45].
lian spermatozoa have the intrinsic ability to bind It should be noted that sperm uptake may not be the
exogenous DNA. In 1989, Spadafora and co-workers, only limiting factor in sperm-mediated gene transfer,
using mice as a model, reported that such DNA binding since it was recently shown that both fresh and frozen-
capacity of sperm could be used to introduce foreign thawed bull sperm can effectively take up foreign DNA
DNA into eggs during fertilization for making [46]. Consequently, efforts have been made to improve
transgenic animals [34]. The report generated sub- the integration of transgenes into the host genome.
stantial interest because   sperm-mediated DNA trans- Shemesh et al. employed the restriction enzyme
fer  was simple and low cost. However, difficulties in mediated insertion method to integrate transgenes into
reproducibility and low efficiency in integrating the bull sperm genome and reported success in
transgenes into the animal genome, resulted in producing transgenic offspring [47]. The mechanism
considerable controversy over the work for several governing foreign DNA integration during sperm-
years [35]. Nevertheless, numerous papers have been mediated gene transfer is not well understood. It has
published in recent years confirming that sperm from been proposed that chromatin sites that are enriched
many species, including livestock, poultry and fish, can with the long interspersed nuclear elements are the
be used as vectors to deliver transgenes into the animal potential sites for foreign DNA integration [48].
genome [36]. Clearly, more work is needed before this method is
Variation in transgenic efficiency has been observed widely applied.
among livestock species in success of sperm-mediated
gene transfer. Very efficient DNA transfer was achieved 4. DNA transfer by somatic cell cloning
in pigs (up to 80% of the offspring were transgenic)
[37], whereas the method was of limited success in Although somatic cell cloning is generally thought of
producing transgenic cattle [38]. The underlying as a method for producing unlimited numbers of
causes for the laboratory-to-laboratory and species- genetically identical offspring, an arguably more
to-species variation that has been observed are still important application is the production of complex
unclear; consequently, sperm-mediated gene transfer genetic modifications in animals. Somatic cell cloning
has not been widely adopted for making transgenic has recently been used to accomplish multiple
animals. modifications in a single cell line, targeted insertions
One approach to improve the efficiency of sperm- and microchromosome transfer.
mediated gene transfer incorporates intracytoplasmic
injection of sperm (ICSI) coated with DNA. Perry 4.1. Sequential genetic modifications
et al. reported that when intact or membrane-disrupted
mouse spermatozoa, incubated with foreign DNA, Because livestock species have long reproduction
were microinjected into eggs by ICSI, about 20% of cycles, breeding is not an ideal approach for generating
the offspring expressed the integrated exogenous DNA homozygous modifications or assembling multiple
[39]. Limited success of ICSI in some species, genetic modifications in a single line. The alternative
however, has impeded application of the technique. is to generate genetic modifications sequentially with-
To our knowledge, success in using ICSI in sperm- out germ line transmission. If immortal embryonic stem
mediated gene transfer has been reported only in pigs cell lines were available for livestock species, multiple
[40 43]. modifications could be made rapidly in vitro. There has
Researchers have also employed electroporation or been substantial effort to develop stems cells in
liposome treatment to facilitate foreign DNA uptake by livestock, with some success, but they have not been
sperm [36]. Recently, a linker-based sperm-mediated generally used for genetic modification. Therefore,
gene transfer method was reported in which DNA was sequential modifications must be done in primary cells
mixed with a monoclonal antibody that recognized a with finite life spans, which is a challenge [49 54]. Only
130 J.M. Robl et al. / Theriogenology 67 (2007) 127 133
one genetic modification can be made in the lifespan of 4.2. Targeted DNA insertion
a cell line because the number of cell divisions needed
to establish a cell line for transfection and selection of In non-murine mammalian species, gene targeting
colonies of transgenic cells for embryonic cloning has been accomplished in primary somatic cells, that
requires nearly the entire cell lifespan. Sequential were then subjected to embryonic cloning to produce
genetic modifications, therefore, require rejuvenation of offspring [6 10]. Because of low recombination
the cell lines by embryonic cloning following each frequency in somatic cells, gene targeting was first
modification. successful with genes that were transcriptionally active
Repeated embryonic cloning may reduce cloning in the cell line used for targeting and was accomplished
efficiency as a result of accumulated epigenetic errors in sheep and pig [6 9]. Transcriptionally active genes
induced by the cloning process. Very little informa- are more amenable to gene targeting, compared to silent
tion is available on the impact of repeated cloning on genes, because they have a higher frequency of
cell viability and developmental potential of clones. homologous recombination [7,49] and selection of
The most extensive report on repeated cloning was correctly targeted cells can be done by having the
conducted in mice [55]. Two lines of mice were targeted gene promoter drive expression of a selection
repeatedly cloned from cumulus cell nuclear donors. marker; so-called   promoter-less  positive selection
One line did not produce offspring beyond four [6 9].
rounds of repeat cloning and the other line produced To fully evaluate the consequences of a genetic
one cannibalized offspring at generation six. Cloning modification, both alleles of the gene must be targeted.
efficiency was low in the first generation but showed a In pigs, two innovative approaches were used for
tendency to decline in subsequence generations in one targeting the second allele of the a (1,3)-galactocyl-
line (from 4.2 to 0.1%). Abnormalities associated transferase gene. Heterozygous KO fibroblasts were
with cloning, such as large placental size, were selected in vitro for the lack of the enzymatic activity
similar for each generation. The offspring did not resulting either from a spontaneous point mutation in
show symptoms of premature ageing or other the second allele of the gene [57] or from mitotic
abnormalities beyond what was normally observed recombinants [58]. Unfortunately, these approaches are
in first generation cloned mice. Serial cloning in neither useful for silent genes nor widely applicable for
cattle was successful up to two rounds of cloning but active genes.
offspring were not produced following a third round Homozygous targeting of a silent gene, the
of cloning [56]. The calf produced from two rounds of immunoglobulin m gene, was recently accomplished
cloning appeared normal and was fertile. Evidence in the cow [10]. The gene was characterized to identify a
from these studies indicated that, although apparently polymorphic marker, outside the targeting sequence to
healthy animals can be made from serial cloning, the distinguish the two alleles (designated A and B). A
efficiency of cloning is reduced to a point where it nonisogenic targeting vector was prepared containing
was not possible to produce additional live offspring. 7.2 and 2.0 kb homologous sequences, a puro positive
Therefore, using serial cloning to rejuvenate cell lines selection marker driven by a mouse PGK promoter, and
for sequential genetic modifications may limit the a transcriptional and translational STOP cassette. The
number of modifications that can be made in a cell vector was electroporated into bovine fetal fibroblast
line. cells that underwent selection for approximately 3
Previously, we reported using serial cloning to weeks. Colonies were divided; one half was used for
rejuvenate genetically modified cell lines [10]. Calves PCR and the other half for embryonic cloning. Cloned
were produced following two and three rounds of fetuses were recovered at 40 days of gestation to
cloning at similar efficiency (8 and 6%, respectively) rederive cell lines. Of the six fetuses recovered, three
demonstrating that a minimum of two sequential were positive by PCR; two had the knockout vector
genetic modifications were compatible with term integrated into allele A and one into allele B.
development of clones (calves derived from a single Rejuvenated cell lines were used to produce cloned
cloning are designated G1, from two rounds of cloning heterozygous targeted calves at a rate of 13 calves from
are G2, etc.). In addition, fetal cell lines were produced 153 recipients (8%).
from five rounds of cloning with four genetic Targeting the second allele of the m immunoglobulin
modifications. Fetuses and fibroblast cell lines appeared gene was more of a challenge because the targeting
normal. In our more recent work, we have produced G5 vector could either replace the first targeting vector or
calves with four genetic modifications. target the second allele. To increase the probability of
J.M. Robl et al. / Theriogenology 67 (2007) 127 133 131
targeting to allele A, the short arm of the targeting Currently, 21 live transchromosomic calves have been
vector was replaced by a sequence directly PCR produced and have been shown to retain the HAC by
amplified from allele A of the cell line. In addition, the G418 selection. Fibroblasts and peripheral blood
positive selection marker was replaced by a neo lymphocytes (PBLs) from four calves were evaluated
cassette. In seven positive colonies derived from allele for HAC retention by fluorescent in situ hybridization.
B heterozygous cell line, six were correctly targeted to Results of FISH analysis indicated that the HAC was
allele A. Cloned fetuses were produced to rederive cell retained as an independent chromosome and the
lines. Rejuvenated cell lines were used to produce proportion of cells retaining the HAC ranged from 78
cloned homozygous targeted calves at a rate of eight to 100%. Some of these calves are now over 3 year old and
calves from 137 recipients (6%). HAC retention rate in PBLs has not changed with age.
These results demonstrated that sequential homo- To determine whether human immunoglobulin loci
zygous targeting was possible for an inactive gene were rearranged and expressed, we performed RT-PCR
using primary fetal fibroblasts and embryonic cloning. analysis on PBLs. We observed the expression of both
Calves were produced at reasonable efficiency follow- human heavy and lambda genes in the PBLs of all the
ing three successive cloning cycles. This method calves and the diversity of the human heavy and lambda
should be useful for producing other types of targeted repertoire was determined by sequence analysis. The
genetic modifications. DNA sequences had a wide utilization of VH/Vl, DH
and JH/Jl segments distributed over the loci. In the
4.3. Microchromosome transfer lambda transcripts, the frequent utilization of JH4 and
of V segments from VH1 and VH3 were detected
Artificial chromosome vectors have the capacity to (similar to humans). Addition of nongerm-line nucleo-
carry very large, Mb-sized, sequences of DNA. tides (N-addition), as well as nucleotide deletion, was
Artificial chromosome vectors are maintained in cells also observed in both heavy and lambda transcripts.
as autonomous, replicating chromosomes. Conse- These produced a high degree of diversification in the
quently, the vector must contain a centromere, two third complementarity-determining region of both the
telomeres and origins of replication. Ishida and cow- heavy and lambda chains. In addition, low levels of
orkers [59 61] have generated human artificial chro- human immunoglobulin protein were detected in
mosome (HAC) vectors from randomly generated precolostrum calves.
chromosome fragments. These HACs were transferred
into mice and some were stable.
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