STATE OF THE ART IN THE PRODUCTION OF TRANSGENIC AND CLONED GOATS
Vicente José de F. Freitas1, Irina A. Serova2, Lyudmila E. Andreeva3 & Oleg L. Serov2
1
Universidade Estadual do Ceará, Laboratório de Fisiologia e Controle da Reprodução, Fortaleza, Brazil
Institute of Cytology and Genetics, Russian Academy of Sciences, Novosibirsk, Russia
3
Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia
2
ABSTRACT
This review summarizes the advances in the field of transgenic goats for the objective of
producing recombinant proteins in their milk. Special attention is done to the results obtained by
our group in the last year. Before these technologies are implemented in goat specific programmes,
efficiencies must be improved, costs reduced, and regulatory approval obtained for the marketing
of products derived from such animals.
Key words: goats, transgenesis, cloning
Running-title: Transgenesis and cloning in goats
I. INTRODUCTION
The production of human recombinant pharmaceuticals in the milk of transgenic farm
animals (Clark, 1998) solves many of the problems associated with microbial reactors (lack of posttranslational modifications, high purifications costs) or animal cell bioreactors (high capital costs,
expensive culture media, low yields). Dairy goats are ideal for transgenic production of therapeutic
recombinant proteins. At concentrations of recombinant protein of 1-5 g/L that have been
reproducibly achieved with various animal models, herds of transgenic goats of manageable size
could easily yield 1-300 kg of purified product per year (Baguisi et al., 1999).
Following the birth of the first cloned sheep (Wilmut et al., 1997), somatic cell nuclear
transfer (SCNT) has been proposed as a method to clone high genetic merit animals and
endangered species. The main utilization of this technology in goats has been the generation of
transgenic founders using in vitro-transfected cell lines with DNA expression vector of interest, and
the cloning of such transgenic animal founders (Keefer et al., 2002; Baldassarre et al., 2003a).
In addition, in August 2006, ATryn®, GTC's recombinant form of human antithrombin, was
approved by the European Commission for use in patients with hereditary antithrombin deficiency
undergoing surgical procedures. This was the first approval anywhere in the world of a therapeutic
protein produced from a transgenic animal.
The present manuscript reviews the methods used for production of transgenic and cloned
goats, as well as the current and potential application of these technologies in Brazil.
II. METHODS FOR TRANSGENIC GOAT PRODUCTION
Pronuclear microinjection and SCTN have been the two methods of choice for producing
transgenic goats. Other methods may be possible in the future, but their application for producing
transgenic goats has not been reported. As example of these methods can be cited sperm-mediated
gene transfer, the use of recombinant viral vectors, the establishment and use of embryonic stem
cells and microprojectile cell bombardment technique (reviewed in Houdebine, 2002).
Following the first report of generating transgenic mice produced by pronuclear
microinjection (Gordon et al., 1980), transgenic rabbits, sheep, pigs, cattle and goats have been
reported thereafter using the same technology. Therefore, the traditional method for producing
transgenic founder goats involves the microinjection of a DNA construct into the pronuclei of in
vivo produce embryos (Ebert et al., 1991; Lee et al., 2000). However, using standard
superovulation protocols the follicles often ovulate within a wide range of hours resulting in
variable stages of development of the embryos collected. It is also accepted that transgenesis rates
are more favorable if microinjection is performed at the early pronuclear stage of development, i.e.
15-20 h after fertilization (Wang & Yang, 2002). Consequently, the ability to recover an evenly
staged group of zygotes is very important for the overall success of a DNA microinjection
programme. Thus, advances in the production of transgenic goats by pronuclear microinjection
have been reported recently by using in vitro produced zygotes from laparoscopic ovum pick-up
(LOPU)-derived oocytes (Baldassarre et al., 2003b). This method increases the number of
procedures performed in the life of donors, is more predictable in terms of the number of embryos
per ova produced, and enables controlling timing of fertilization and, subsequently, DNA
microinjection, which is a critical factor in successful integration.
The SCNT using transgenic donor cells is an efficient means for generation of transgenic
founder goats, especially in regard to the number of animals required to produce a transgenic
founder expressing the protein of interest (Baldassarre et al., 2003a). Donor cells can be selected
for gender, genetically modified to introduce the gene of interest, and screened for incorporation of
the transgene into the genome, including copy number and integration site, before use in SCNT.
A number of cell types have been used as donor cells for SCNT. In goats, fetal fibroblasts
are generally the cell type of choice for production of transgenic cell lines; however, other cells
types, including granulose cells and skin fibroblasts cells obtained from adult animals have been
used (Lazaris et al., 2006).
The incidence of perinatal loss associated with SCNT has not been reported in the goat,
although this lack of information may be due to (a) the relatively low number of goat clones
produced to date or (b) a minimal in vitro culture period in which reconstructed embryos were
transferred at the two- to four-cell stage in previous studies. However, Yong & Yuqiang (1998)
produced an amazing 45 cloned goats from the transfer of 141 serially reconstructed embryos into
29 recipients in a cloning study using blastomere donor nuclei, in which embryos were cultured up
to the morula stage prior to transfer. Thus, manipulated goat embryos may not be as sensitive as
cow and sheep embryos to micromanipulation procedures and in vitro culture conditions.
III. OUR RESULTS
Following production of transgenic mice that secrete high levels of human Granulocyte
Colony Stimulating Factor (hG-CSF) into their milk (Dvoryanchikov et al., 2005) at the Instituto de
Biofísica Carlos Chagas Filho (IBCCF, Rio de Janeiro, Brazil), we initiated a project to produce
transgenic goats to hG-CSF. This protein was chosen due to its importance to the human health.
The hG-CSF is a hematopoietic growth factor that stimulates the proliferation and the
differentiation commitment of neutrophil precursor cells, and enhances some of the functional
properties of mature neutrophils (Morstyn & Burgess 1988). Following its production as a
recombinant human protein, hG-CSF has been the most widely used hematopoietic growth factor
due to its proven efficacy against different forms of neutropenia, chemotherapy induced leucopenia,
and in the mobilization of progenitor cells for autologous or allogenic transplantation.
Thus, experiments were performed in the Laboratório de Fisiologia e Controle da
Reprodução (LFCR, Fortaleza, Brazil) using Saanen goats as donors and undefined breed goats as
recipients of microinjected embryos. The first experiments established the basis of synchronization
and superovulation regimen for the purpose of collecting and manipulating the pronuclear embryo
(Freitas et al., 2003). These experiments showed that microinjected pronuclear goat embryos can
survive to produce live kids following manipulation, indicating that the project designed to produce
transgenic goats is feasible.
Donors and recipients were synchronized by use of a progestagen treatment during 11 days.
Superovulation was achieved in donors after an FSH regimen with six decreasing doses. The
fertilization was provided by Saanen bucks with proven fertility and the presumable zygotes were
recovered approximately 72 h after progestagen removal by flushing of oviducts. The recovered
zygotes were briefly centrifuged to a reliable visualization of the pronuclei. The DNA construction
containing hG-CSF gene flanked by goat and bovine αs1-casein genes was injected into pronuclei
using an inverted microscopy with micromanipulators and Nomarski optics (Figure 1).
Figure 1. A goat zygote after centrifugation with two visible pronuclei (A) and the same zygote
during microinjection (B, x300).
Successfully microinjected embryos were transferred into the oviduct of synchronized
recipient goats following laparoscopic exploration to confirm the presence of at least one recent
ovulation. For embryo transfer, a mid-ventral laparotomy was established and the reproductive tract
was exteriorized. A Tomcat catheter containing the embryos was introduced through the fimbria
into the oviduct and the embryos were injected into the lumen. Three to nine embryos were
transferred per recipient. Pregnancy was detected by ultrasound at 28–35 days following transfer
and the pregnant recipients were monitored until the kidding. Finally, the kids were screened by
PCR analysis (Table 1), using genomic DNA extracted from the ears of two-week-old kids
according to the method indicated by Sambrook et al. (1989).
Table 1. Summary of results obtained after several experiments with goat transgenesis in
Northeastern Brazil.
Order
of experiment
01
02
03
donors
12
17
23
emb. microinjected
85
90
129
Nb. of
recipients
17
20
27
kids born
2
14
12
transgenics
0
0
1
IV. CONCLUSIONS
The efficiencies in the production of transgenic goats have been dramatically improved in
the last few years. The efficiencies have been sufficient to allow a few groups to produce
recombinant proteins of pharmaceutical and biomedical interest, for commercial applications. In
addition, cloning has the potential to improve the efficiency of transgenesis in these applications.
Finally, the result obtained by our group was the first report of birth of transgenic goat in
Latin America and represents a great step for the use of this technology in Brazil.
ACKNOWLEDGMENTS
Authors are grateful to FINEP (Rio de Janeiro), BNB/FUNDECI (Fortaleza) and CNPq
(Brasília) for funding. Special thanks go to the LFCR team for taking great care of the animals.
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