Skip to main content
Download PDF

Specific genetic modifications of domestic animals by gene targeting and animal cloning

Reproductive Biology and Endocrinology volume 1, Article number: 103 (2003) Cite this article

Abstract

The technology of gene targeting through homologous recombination has been extremely useful for elucidating gene functions in mice. The application of this technology was thought impossible in the large livestock species until the successful creation of the first mammalian clone "Dolly" the sheep. The combination of the technologies for gene targeting of somatic cells with those of animal cloning made it possible to introduce specific genetic mutations into domestic animals. In this review, the principles of gene targeting in somatic cells and the challenges of nuclear transfer using gene-targeted cells are discussed. The relevance of gene targeting in domestic animals for applications in bio-medicine and agriculture are also examined.

Introduction

Discovery of the functions of genes is a remarkably important aspect of biological research. Precise genetic modifications of animals are a powerful methodology to study physiological mechanisms at the molecular level. Gene knock-in and knock-out through gene targeting mediated by DNA homologous recombination paved the way towards the era of mammalian functional genomics. Following the first report of mouse embryonic stem (ES) cells in 1981 [1, 2] and the successful alteration of the hypoxanthine phosphoribosyl transferase (HPRT) gene locus through homologous recombination in mouse ES cells in 1989 [3, 4], numerous mouse mutants generated through gene targeting have been reported. By the year 2000, an estimated 5000 genes had been inactivated in the mouse [5]. It has become routine in many laboratories around the world to produce mice with specific genetic modifications including gene disruption, gene replacement, and even engineered chromosomal translocation. However, it has been extremely difficult to alter genes in mammals, other than the mouse, by homologous recombination. Although several ES cell lines have been established, primarily from the 129/SvJ mouse strain, and are now commercially available, cloning of ES cells from other mammalian species has made only limited progress. Embryo-derived pluripotent cell lines have been reported in pigs [6] and cattle [7], but successful germ-line chimeric offspring have not yet been reported. The failure to development of ES cell lines has hampered many applications of gene targeting technologies in domestic animals.

The breakthrough in animal cloning using somatic cells [8] implies that targeted genetic manipulations of domestic animals could be achieved by combining gene targeting in somatic cells and cloning. In this review, the strategy and potential challenges of gene targeting manipulation in the domestic animal are discussed. Some of the applications of gene targeting in domestic animals for bio-medicine and agriculture are also addressed.

Homologous recombination and gene targeting

Gene targeting is the terminology used for genetic manipulations of animal genomes using homologous recombination for the altering of gene activity in any purposeful manner. Homologous recombination is the exchange of homologous segments of two DNA molecules anywhere along their length. The technology of homologous recombination allows the precise modification (replacement or deletion) of certain alleles in the genome. In mammals, homologous recombination naturally occurs during the meiosis cleavage of gamete cells. Two homologous chromosomes undergo a crossing-over process at recombinant hot areas, resulting in the exchange of chromosomal fragments. A primary step in homologous recombination is DNA exchange, which involves pairing of a DNA duplex with at least one DNA strand containing a complementary sequence to form an intermediate recombinant structure. When two complementary DNA strands pair with a DNA duplex, a classical Holliday recombination-joint may form [9]. Once formed, a hetero-duplex structure may be resolved by strand breakage and exchange, so that all or a portion of an invading DNA strand is spliced into a recipient DNA duplex, adding or replacing a segment of the recipient DNA.

The majority of foreign DNA integration events in yeast are homologous recombination [10] as opposed to random illegitimate recombination in mammalian cells [11]. The research on DNA transformation in the 1970s in yeast led to the development of the basic principles of gene disruption via homologous recombination in mammalian cells. The insertion of DNA sequences into a human chromosome by homologous recombination was first demonstrated in 1985 by Smithies et al. [12] for the human β-globulin locus. Later, Capacchi's work in 1989 [13] laid the foundation for the development of the strategies of gene targeting using mouse ES cells.

The most commonly used targeting vector in mouse ES cells is the replacement vector (Fig. 1), which contains a positive selection marker for selection of transformed cells. Additionally, a negative selection marker is added outside of the region of the homology to counter-select the random integration following positive selection. During homologous recombination, the negative selectable gene is lost because it is located at the distal region of homology between a vector and a target. Therefore, the cells containing negative selection markers are resulted from random DNA integrations. Usually, the simplex virus-thymidine kinase (HSV-tk) gene is used for negative selection. The cells with the HSV-tk gene are sensitive to the nucleotide analogs gancyclovir or 1-(2-deoxy-2-fluoro-beta-D-arabino-furanosyl)-5-iodouracil (FIAU). As a result, only the cells transformed through homologous recombination are isolated following negative selection [13]. This procedure is termed positive-negative selection (PNS). The ES cells with targeted mutant genes can then be injected into host blastocysts to derive chimeric mice. Eventually, some of the chimeric mice could transmit the genotype of the ES cells to their progeny.

Figure 1
figure 1

The replacement vector for gene targeting. The third exon of a target gene is disrupted by neomycin resistance marker (Neo-Res) after homologous recombination. The HSV thymidine kinase gene (HSV-tk) is removed following homologous recombination.

Full size image

Gene targeting in somatic cells and animal cloning

The general strategy of gene targeting using animal cloning

The first animal cloned via nuclear transfer of an adult somatic cell, Dolly the sheep [8], sparked vast interest in nuclear reprogramming of somatic cells and animal cloning. Nuclear transfer technology is now widely used by numerous laboratories to produce animals from the somatic cells of animal fetuses and adults. In the last several years, the procedures of nuclear transfer have been successfully applied to various mammalian species and have resulted in live births of mice [14], cattle [15], goats [16, 17], pigs [18], rabbits [19] and cats [20]. Furthermore, transgenic animals have been produced by cloning gene-transfected fetal somatic donor cells in sheep [21], cattle [22] and goats [17]. A procedure for producing cloned animals through nuclear transfer of gene-targeted somatic cells is shown in Fig. 2. Somatic cells (usually fetal fibroblastic cells) are transfected in vitro by liposomes or electroporation, screened for gene targeting, and transferred into enucleated oocytes. The couplets are electrically pulsed to induce fusion and then activated for further development in vitro. The embryos derived from nuclear transfer are transferred into recipients which give birth to offspring carrying mutations from the gene-targeted somatic cells. Since cloning of pluripotent ES cells in domestic animals has not been fully demonstrated, to date, it has not been possible to knock-in or knock-out genes in domestic animals by the homologous recombination in ES cells and the production of animal chimeras. Alternatively, the technology of animal cloning might provide a basis for gene targeting in farm animals. In fact, gene-targeted animal clones have been successfully produced in sheep [23] and pigs [24] by homologous recombination in fetal fibroblast cells and nuclear transfer.

Figure 2
figure 2

Procedure of producing gene-targeted animals (goats) by cloning.

Full size image

Gene targeting in somatic cells

A number of parameters have been shown to influence the frequency of homologous recombination in mouse ES cells: (1) the frequency increases when longer homologous sequences are included in the targeting constructs [25]; (2) homologous recombination with isogenic DNA sequences is more efficient than with non-isogenic sequences [26]; (3) absolute frequency of homologous recombination appears to be locus dependant [27]. The absolute targeting frequency in mouse ES cells varies from 1 × 10-5 to 1 × 10-6 per electroporated cell [28]. However, accumulated evidence shows that gene targeting in somatic cells is more difficult than in mouse ES cells, and the frequency in somatic cells is about two orders of magnitude lower than in mouse ES cells [29], for example, ranging from 2.8 × 10-7 to 27.5 × 10-7 per cell in sheep [30]. Apparently, mouse ES cells possess higher recombinogenicity than somatic cells. Therefore, a more efficient strategy for promoting homologous recombination is required for gene targeting in somatic cells.

Although PNS for mouse ES cells can yield an enrichment of about 2000 fold [13], PNS in somatic cells can only achieve a 2–3 fold enrichment [12]. A powerful promoterless selection has been employed for increasing enrichment of gene targeting events in somatic cells. In a promoterless vector, the gene of the positive selection marker lacks a promoter, it can only be expressed from the internal promoter of the target gene following homologous recombination. In some very rare cases, non-homologous recombination events could still be recovered if the targeting vectors integrate into the sites of chromosomes close to an active promoter. Hanson et al. [12] reported that an enrichment ratio of 5,000- to 10,000-fold with a promoterless vector in a rat fibroblastic cell line for the c-myc gene had been achieved. In a report of generating α (1,3) galactosyltransferase knock-out porcine fetal fibroblasts, it was also demonstrated that the promoterless targeting vector provided a significant enhancement in gene targeting efficiency by eliminating a larger proportion of random integration events in comparison to the PNS strategy [31].

The somatic cell lines selected for gene targeting and nuclear transfer must have high potency for population expansion and a high stability of cytogenetic normalcy. Theoretically, a total of ~45 population doublings are required to generate gene-targeted cells for nuclear transfer [32]. An obvious choice among the somatic cell types is the fetal fibroblast cell due to its proved ability to produce cloned animals and the ease of population expansion. The life span of the fetal fibroblast is about 30–50 population doublings in pigs and cattle [33] and 40–120 population doublings in sheep [30]. Therefore, it is possible to carry out gene targeting procedures with these cells. Furthermore, it is feasible to introduce targeted mutations into animals through nuclear transfer, since evidence shows that populations of bovine fibroblasts, after more than 45 cell doublings [34], or even in senescence [35], retained their totipotency for supporting full development of cloned embryos. Up to now, several reports of gene-targeted modifications in sheep [23, 36] and pigs [24, 37] using promoterless targeting vectors and fetal fibroblastic cell lines have been published.

When somatic cells nearing senescence are used, chromosomal abnormality is the critical factor related to poor efficiency of animal cloning. In sheep, telomeres degrade at a rate of ~0.59 kb per year in vivo, but much faster in culture [38]. When telomeres fall below a critical length, chromosomal abnormalities occur more frequently. Somatic cells can be induced to prolong their longevity and retain cytogenetic normalcy after a large number of population doublings. Previous reports indicate that human diploid fibroblasts can be immortalized by transfection with the telomerase catalytic component. With this treatment, the human fibroblasts could be cultured beyond 300 passages without phenotypic or chromosomal abnormalities [39–41]. Therefore, the expression of telomerase in targeted somatic cells could be an effective treatment for improving the efficiency of animal cloning.

Applications of gene targeting in domestic animals

Xenotransplantation

Organ transplantation is the only known treatment for many diseases involving terminal organ failure. Unfortunately, the lack of availability of human organs has greatly limited the number of patients who can receive this life-saving treatment. Because of the serious shortage of human organs, tremendous effort has been made worldwide to the study for the use of genetically engineered animal organs as replacement organs for human transplantation (Xenotransplantation).

Pigs are considered to be the most suitable animal for supplying organs because their organs are physiologically and anatomically compatible with those of humans [42], and they are already a production animal slaughtered for food. Furthermore, pigs can be easily bred, they are very prolific, and can be housed in pathogen-free facilities to prevent the transmission of infectious diseases [43]. One of the major constrains to using pig organs for xenotransplasntation is complement-dependant hyper-acute rejection (HAR), which occurs within a few minutes following tissue transplantation between discordant species of animals. Hyper-acute rejection results in lyses of the endothelial cells in the vessels of the transplanted organs and ultimately in graft failure [43]. Human and old world monkeys have natural antibodies against α (1, 3)-galactosyl epitopes on pig cells. Therefore, the best way to circumvent HAR is to knock-out the function of the α-1, 3-galactosyltransferase gene in the pigs. Mice with an α-1, 3-galactosyltransferase gene inactivated through homologous recombination are viable, and when human serum bound to cells and tissues of these mice they showed substantially less xeno-antibody production than normal mice [44]. One allele of the α-1, 3-galactosyltransferase locus in the pig was successfully knocked out through homologous recombination in somatic cells and subsequent nuclear transfer [24, 37, 45]. More recently, the second allele of α-1, 3-galactosyltransferase gene has also been inactivated by selection of a point mutation at the second base of exon 9, which resulted in inactivation of the gene. Four healthy piglets with double knock-out of α-1, 3-galactosyltransferase gene were produced by nuclear transfer [46]. Further analysis of the tissues and organs from these double knock-out pigs in nonhuman primate models will provide crucial information regarding the mechanisms of HAR.

Knock-out of Prnp gene

Prion diseases have had a devastating impact on the production of livestock, bovine spongiform encephalopathy (BSE, one type of prion disease in bovine) is expected to cause about $50 billion loss in UK. Now, it has been ascertained that one variant of deadly Creuzfeldt Jacob disease (CJD) in humans can be caused by consumption of animal products contaminated with the BSE pathogen [47]. Control of the BSE epidemic has become top priority worldwide.

Animal prion diseases include scrapie of sheep and goats, transmissible mink encephalopathy, chronic wasting disease of mule deer and elk, feline spongiform encephalopathy and BSE. The characteristics of these diseases are infectious and vacuolar degeneration of the gray matter neuropil. It was discovered by Prusiner in 1982 [48] that the infectious agent was a protease-resistant protein, which was termed a prion (PrP). A prion has a molecular weight of 27–30 kDa and is encoded by a single copy gene, Prnp in mammals. Prions exist in two major isoforms: the nonpathogenic or cellular form, designated PrPC, and the pathogenic or scrapie-inducing form, designated PrPSc. Prion diseases are caused by conversion of PrPC to PrPSc through infection or genetic mutation. Once a cell contains PrPSc, it appears to act as a conformational template by which PrPC is converted to a new molecule of PrPSc through protein-protein interactions [49].

Some evidence shows that normal prions play a role in synaptic function [50] and copper binding [51]. However, no developmental or behavioral abnormalities were found in prion knock-out mice. Furthermore, in the prion knock-out mice, homozygous null mice (Prnp0/0) fail to develop the characteristic clinical and neuropathological symptoms of scrapie after inoculation with mouse prions, and they do not propagate prion infectivity [52, 53]. Therefore, it is expected that removal of the Prnp gene from cattle and sheep will result in these animals being resistant to BSE and scrapie. Fetal fibroblast cell lines with the deletion of one allele of the PrP gene have been established in sheep by homologous recombination [30]. Four cloned lambs were produced from nuclear transfer of the PrP +/- somatic cells [36]. More investigations are needed to evaluate the survivability of PrP0/0 domestic animals and their resistance to prion diseases.

Inactivation of animal Ig genes

Human antibodies have numerous diagnostic and therapeutic applications. Current antibody production platforms used in industry are not expected to meet future demand for production capacity. Some of the strategies for mass production of human antibodies involve: (1) introducing the human immunoglobulin gene into mice or large domestic animals (e.g. cattle) with their endogenous immunoglobulin gene inactivated, (2) producing human monoclonal antibodies from mouse hybridoma or polyclonal antibodies from large animal blood immunized against the human antigen. To inactivate the endogenous immunoglobulin gene, gene disruption by homologous recombination in mouse ES cells, or somatic cells of large domestic animals, must be carried out in order to introduce the mutation into the animals through cloning.

Each antibody molecule consists of two classes of polypeptide chains, light (L) chains (κ L-chain or λ L-chain) and heavy (H) chains. A single antibody molecule has two identical copies of the L chain and two of the H chain. The loci of antibodies are very large and located on different chromosomes (chromosome 14 (H-chain locus), 22 (λ L-chain locus) and 2 (κ L-chain) in humans). In their germ line configuration, these loci consist of diverse segments encoding the variable (V), diversity (D) and joining (J) genes that comprise the variable domains along with the segments that encode the constant (C) domains [54].

Mouse antibody is immunogenic in humans. The major factors contributing to this immunogenicity are thought to be the sequences of the murine C- and V-regions of an antibody molecule [55]. Immunogenicity of therapeutic antibodies is a significant problem and severely limits their widespread and repeated applications to treat many diseases. A process of antibody humanization has been employed to structure the mouse antibodies within the framework of the human antibody molecule, while retaining the mouse antigen-binding complementary determining regions [56]. Although partially humanized antibodies have demonstrated significantly reduced immunogenicity, the most desirable antibodies for therapeutic applications in humans would be fully humanized antibodies [57]. Transgenic mice have been constructed which have had their own immunoglobulin genes functionally replaced with human immunoglobulin genes so that they produce human antibodies upon immunization [58–60]. Elimination of mouse antibody production was achieved by inactivation of mouse Ig genes in the ES cells by using gene-targeting technology to delete crucial cis-acting sequences involved in the process of mouse Ig gene rearrangement and expression. B cell development in these mutant mice could be restored by the introduction of megabase-sized YACs containing a human germline-configuration H- and κ L-chain minilocus transgene. The expression of fully human antibody in these transgenic mice was predominant, at a level of several 100 μg /l of blood [60]. This level of expression is several hundred-fold higher than that detected in wild-type mice expressing the human Ig gene [60], indicating the importance of inactivating the endogenous mouse Ig G genes in order to enhance human antibody production by mice. More recently, a human artificial chromosome (HAC) vector containing the entire unarranged sequences of the human Ig H-chain and κ L-chain was successfully introduced into cows (TC cows) with the technology of microcell-mediated chromosome transfer and nuclear transfer of bovine fetal fibroblast cells [61]. The HAC was retained in calves at a rate of 78–100% of cells. Human immunoglobulin proteins were detected in the blood of new born calves at a level of 13–258 μg/l. This report suggests that production of a specific human polyclonal antibody could be easily scaled up by direct immunization of TC cows. Nevertheless, greater challenges remain for increasing expression levels of the human antibodies by inactivation of the endogenous bovine Ig genes. With further improvements to gene targeting technology in somatic cells and nuclear transfer, immunologically deficient cattle or other large domestic animals will be produced in order to provide a platform for expressing various human Ig genes.

Other applications of gene targeting in domestic animals

Gene targeting in domestic animals has other potential applications in both agriculture, and as human disease models. In animal production, the candidate genes for targeting modifications may include: (1) Myostatin gene, which is involved in the expression of the 'double muscle' phenotype of some breeds of cattle [62], it has been suggested that knocking-out this gene would increase skeletal muscle growth in animals [32]; (2) Growth hormone gene, early studies of pigs transgenic for an exogenous growth hormone gene showed an increased growth rate, however poor control of its expression caused many deleterious side effects. Knock-in with extra copies of the growth hormone gene should result in an elevated level of growth hormone with a physiological pattern of release, which may lead to an enhancement of the animal growth rate without the negative effects. Gene targeting has been extensively used in mice to create disease models for studying the pathologies of and therapies for human diseases. However, in some disease models, knock-out mice may exhibit the biochemical pathways of the pathology, but often do not produce clinical symptoms [63]. Genetic engineering of livestock through gene targeting could create better models for human diseases due to greater similarities in anatomy, physiology and organ size. For example, the mouse model of human cystic fibrosis was generated by disruption of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, however, the utility of the CFTR-knock-out mice in studying the pathogenesis of cystic fibrosis has been limited because of their failure, despite the presence of severe intestinal disease, to develop lung disease [64, 65] due to differences in lung physiology. There are substantial similarities between humans and sheep in lung physiology, anatomy, DNA sequence of the CFTR gene, and tissue specific expression patterns of the CFTR gene [66]. Therefore, a knock-out of the CFTR gene in sheep could produce a better model for human cystic fibrosis.

Conclusions

The combined technologies of gene targeting in somatic cells and animal cloning permit the introduction of specific genetic modifications into the genome of domestic animals. Although gene targeting in mouse ES cells has become a routine procedure in many laboratories worldwide, the efficiency of gene targeting in somatic cells is still very low. For some genes, it is even impossible to implement the strategy of gene targeting using promoterless targeting vectors, due to the lack of active promoters in fibroblasts and other types of somatic cells. Nevertheless, continuous research efforts aimed at cloning stem cells of domestic animals might eventually provide a platform for efficient gene targeting using the technology similar to that for mouse ES cells. The application of gene targeting technologies in domestic animals for xenotransplantation has made significant progress in recent years. Generation of homozygous knock-out pigs for the α-1, 3-galactosyltransferase gene will eventually elucidate the mechanism(s), and lead to the prevention of hyper-acute immuno-rejection of transplanted tissues and organs. In the foreseeable future, gene targeting will likely play a major role in the manufacture of pharmaceuticals, and comprise much of the research examining physiological mechanisms as well as analyzing gene functions in domestic animals.

References

  1. Evans MJ, Kaufman MH: Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981, 292: 154-156.

    CAS  Article  PubMed  Google Scholar 

  2. Martin GR: Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A. 1981, 78: 7634-7638.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  3. Doetschman T, Gregg RG, Maeda N, Hooper ML, Melton DW, Thompson S, Smithies O: Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature. 1987, 330: 576-578.

    CAS  Article  PubMed  Google Scholar 

  4. Thomas KR, Capecchi MR: Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 1987, 51: 503-512.

    CAS  Article  PubMed  Google Scholar 

  5. Houdebine LM: The methods to generate transgenic animals and to control transgene expression. J Biotechnol. 2002, 98: 145-160.

    CAS  Article  PubMed  Google Scholar 

  6. Wheeler MB: Development and validation of swine embryonic stem cells: a review. Reprod Fertil Dev. 1994, 6: 563-568.

    CAS  Article  PubMed  Google Scholar 

  7. Stice SL, Strelchenko NS, Keefer CL, Matthews L: Pluripotent bovine embryonic cell lines direct embryonic development following nuclear transfer. Biol Reprod. 1996, 54: 100-110.

    CAS  Article  PubMed  Google Scholar 

  8. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH: Viable offspring derived from fetal and adult mammalian cells. Nature. 1997, 385: 810-813.

    CAS  Article  PubMed  Google Scholar 

  9. DNA Replication, Repair, and Recombination. In Molecular Cell Biology. Edited by: Harvey L, David B, Arnola B, Lawrence Z, Paul M, James D. 1997, New York: W.H. Freeman and Company, 365-404. 3

  10. Schiestl RH, Petes TD: Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1991, 88: 7585-7589.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  11. Bishop JO, Smith P: Mechanism of chromosomal integration of microinjected DNA. Mol Biol Med. 1989, 6: 283-298.

    CAS  PubMed  Google Scholar 

  12. Hanson KD, Sedivy JM: Analysis of biological selections for high-efficiency gene targeting. Mol Cell Biol. 1995, 15: 45-51.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  13. Capecchi MR: The new mouse genetics: altering the genome by gene targeting. Trends Genet. 1989, 5: 70-76.

    CAS  Article  PubMed  Google Scholar 

  14. Wakayama T, Perry AC, Zuccotti M, Johnson KR, Yanagimachi R: Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature. 1998, 394: 369-374.

    CAS  Article  PubMed  Google Scholar 

  15. Kato Y, Tani T, Sotomaru Y, Kurokawa K, Kato J, Doguchi H, Yasue H, Tsunoda Y: Eight calves cloned from somatic cells of a single adult. Science. 1998, 282: 2095-2098.

    CAS  Article  PubMed  Google Scholar 

  16. Baguisi A, Behboodi E, Melican DT, Pollock JS, Destrempes MM, Cammuso C, Williams JL, Nims SD, Porter CA, Midura P, et al: Production of goats by somatic cell nuclear transfer. Nat Biotechnol. 1999, 17: 456-461.

    CAS  Article  PubMed  Google Scholar 

  17. Keefer CL, Baldassarre H, Keyston R, Wang B, Bhatia B, Bilodeau AS, Zhou JF, Leduc M, Downey BR, Lazaris A, et al: Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes. Biol Reprod. 2001, 64: 849-856.

    CAS  Article  PubMed  Google Scholar 

  18. Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, Dai Y, Boone J, Walker S, Ayares DL, et al: Cloned pigs produced by nuclear transfer from adult somatic cells. Nature. 2000, 407: 86-90.

    CAS  Article  PubMed  Google Scholar 

  19. Chesne P, Adenot PG, Viglietta C, Baratte M, Boulanger L, Renard JP: Cloned rabbits produced by nuclear transfer from adult somatic cells. Nat Biotechnol. 2002, 20: 366-369.

    CAS  Article  PubMed  Google Scholar 

  20. Shin T, Kraemer D, Pryor J, Liu L, Rugila J, Howe L, Buck S, Murphy K, Lyons L, Westhusin M: A cat cloned b y nuclear transplantation. Nature. 2002, 415: 859-

    CAS  Article  PubMed  Google Scholar 

  21. Schnieke AE, Kind AJ, Ritchie WA, Mycock K, Scott AR, Ritchie M, Wilmut I, Colman A, Campbell KH: Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science. 1997, 278: 2130-2133.

    CAS  Article  PubMed  Google Scholar 

  22. Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce de Leon FA, Robl JM: Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science. 1998, 280: 1256-1258.

    CAS  Article  PubMed  Google Scholar 

  23. McCreath KJ, Howcroft J, Campbell KH, Colman A, Schnieke AE, Kind AJ: Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature. 2000, 405: 1066-1069.

    CAS  Article  PubMed  Google Scholar 

  24. Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, Samuel M, Bonk A, Rieke A, Day BN, et al: Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science. 2002, 295: 1089-1092.

    CAS  Article  PubMed  Google Scholar 

  25. Deng C, Capecchi MR: Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol Cell Biol. 1992, 12: 3365-3371.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  26. te Riele H, Maandag ER, Berns A: Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. Proc Natl Acad Sci U S A. 1992, 89: 5128-5132.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  27. Yanez RJ, Porter AC: A chromosomal position effect on gene targeting in human cells. Nucleic Acids Res. 2002, 30: 4892-4901.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  28. Templeton NS, Roberts DD, Safer B: Efficient gene targeting in mouse embryonic stem cells. Gene Ther. 1997, 4: 700-709.

    CAS  Article  PubMed  Google Scholar 

  29. Sedivy JM, Dutriaux A: Gene targeting and somatic cell genetics – a rebirth or a coming of age?. Trends Genet. 1999, 15: 88-90.

    CAS  Article  PubMed  Google Scholar 

  30. Denning C, Dickinson P, Burl S, Wylie D, Fletcher J, Clark AJ: Gene targeting in primary fetal fibroblasts from sheep and pig. Cloning Stem Cells. 2001, 3: 221-231.

    CAS  Article  PubMed  Google Scholar 

  31. Harrison SJ, Guidolin A, Faast R, Crocker LA, Giannakis C, D'Apice AJ, Nottle MB, Lyons I: Efficient generation of alpha(1,3) galactosyltransferase knockout porcine fetal fibroblasts for nuclear transfer. Transgenic Res. 2002, 11: 143-150.

    CAS  Article  PubMed  Google Scholar 

  32. Clark AJ, Burl S, Denning C, Dickinson P: Gene targeting in livestock: a preview. Transgenic Res. 2000, 9: 263-275.

    CAS  Article  PubMed  Google Scholar 

  33. Polejaeva IA, Campbell KH: New advances in somatic cell nuclear transfer: application in transgenesis. Theriogenology. 2000, 53: 117-126.

    CAS  Article  PubMed  Google Scholar 

  34. Kubota C, Yamakuchi H, Todoroki J, Mizoshita K, Tabara N, Barber M, Yang X: Six cloned calves produced from adult fibroblast cells after long-term culture. Proc Natl Acad Sci U S A. 2000, 97: 990-995.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  35. Lanza RP, Cibelli JB, Blackwell C, Cristofalo VJ, Francis MK, Baerlocher GM, Mak J, Schertzer M, Chavez EA, Sawyer N, et al: Extension of cell life-span and telomere length in animals cloned from senescent somatic cells. Science. 2000, 288: 665-669.

    CAS  Article  PubMed  Google Scholar 

  36. Denning C, Burl S, Ainslie A, Bracken J, Dinnyes A, Fletcher J, King T, Ritchie M, Ritchie WA, Rollo M, et al: Deletion of the alpha(1,3)galactosyl transferase (GGTA1) gene and the prion protein (PrP) gene in sheep. Nat Biotechnol. 2001, 19: 559-562.

    CAS  Article  PubMed  Google Scholar 

  37. Dai Y, Vaught TD, Boone J, Chen SH, Phelps CJ, Ball S, Monahan JA, Jobst PM, McCreath KJ, Lamborn AE, et al: Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs. Nat Biotechnol. 2002, 20: 251-255.

    CAS  Article  PubMed  Google Scholar 

  38. Shiels PG, Kind AJ, Campbell KH, Waddington D, Wilmut I, Colman A, Schnieke AE: Analysis of telomere lengths in cloned sheep. Nature. 1999, 399: 316-317.

    CAS  Article  PubMed  Google Scholar 

  39. Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Harley CB, Shay JW, Lichtsteiner S, Wright WE: Extension of life-span by introduction of telomerase into normal human cells. Science. 1998, 279: 349-352.

    CAS  Article  PubMed  Google Scholar 

  40. Szabo S, Folkman J, Vattay P, Morales RE, Pinkus GS, Kato K: Accelerated healing of duodenal ulcers by oral administration of a mutein of basic fibroblast growth factor in rats. Gastroenterology. 1994, 106: 1106-1111.

    CAS  PubMed  Google Scholar 

  41. Jiang XR, Jimenez G, Chang E, Frolkis M, Kusler B, Sage M, Beeche M, Bodnar AG, Wahl GM, Tlsty TD, et al: Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nat Genet. 1999, 21: 111-114.

    CAS  Article  PubMed  Google Scholar 

  42. Ye Y, Niekrasz M, Kosanke S, Welsh R, Jordan HE, Fox JC, Edwards WC, Maxwell C, Cooper DK: The pig as a potential organ donor for man. A study of potentially transferable disease from donor pig to recipient man. Transplantation. 1994, 57: 694-703.

    CAS  Article  PubMed  Google Scholar 

  43. Yang X, Tian XC, Dai Y, Wang B: Transgenic farm animals: applications in agriculture and biomedicine. Biotechnology annual review. 2000, 5: 269-292.

    CAS  Article  PubMed  Google Scholar 

  44. Tearle RG, Tange MJ, Zannettino ZL, Katerelos M, Shinkel TA, Van Denderen BJ, Lonie AJ, Lyons I, Nottle MB, Cox T, et al: The alpha-1,3-galactosyltransferase knockout mouse. Implications for xenotransplantation. Transplantation. 1996, 61: 13-19.

    CAS  Article  PubMed  Google Scholar 

  45. Ramsoondar JJ, Machaty Z, Costa C, Williams BL, Fodor WL, Bondioli KR: Production of {alpha}1,3-Galactosyltransferase-Knockout Cloned Pigs Expressing Human {alpha}1,2-Fucosylosyltransferase. Biol Reprod. 2003, 2: 2-

    Google Scholar 

  46. Phelps CJ, Koike C, Vaught TD, Boone J, Wells KD, Chen SH, Ball S, Specht SM, Polejaeva IA, Monahan JA, et al: Production of alpha 1,3-galactosyltransferase-deficient pigs. Science. 2003, 299: 411-414.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  47. Scott MR, Will R, Ironside J, Nguyen HO, Tremblay P, DeArmond SJ, Prusiner SB: Compelling transgenic evidence for transmission of bovine spongiform encephalopathy prions to humans. Proc Natl Acad Sci U S A. 1999, 96: 15137-15142.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  48. Prusiner SB: Novel proteinaceous infectious particles cause scrapie. Science. 1982, 216: 136-144.

    CAS  Article  PubMed  Google Scholar 

  49. DeArmond SJ, Bouzamondo E: Fundamentals of prion biology and diseases. Toxicology. 2002, 181–182: 9-16.

    Article  PubMed  Google Scholar 

  50. Collinge J, Whittington MA, Sidle KC, Smith CJ, Palmer MS, Clarke AR, Jefferys JG: Prion protein is necessary for normal synaptic function. Nature. 1994, 370: 295-297.

    CAS  Article  PubMed  Google Scholar 

  51. Brown DR, Qin K, Herms JW, Madlung A, Manson J, Strome R, Fraser PE, Kruck T, von Bohlen A, Schulz-Schaeffer W, et al: The cellular prion protein binds copper in vivo. Nature. 1997, 390: 684-687.

    CAS  Article  PubMed  Google Scholar 

  52. Prusiner SB, Groth D, Serban A, Koehler R, Foster D, Torchia M, Burton D, Yang SL, DeArmond SJ: Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies. Proc Natl Acad Sci U S A. 1993, 90: 10608-10612.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  53. Bueler H, Raeber A, Sailer A, Fischer M, Aguzzi A, Weissmann C: High prion and PrPSc levels but delayed onset of disease in scrapie-inoculated mice heterozygous for a disrupted PrP gene. Mol Med. 1994, 1: 19-30.

    CAS  PubMed  Google Scholar 

  54. Robl JM, Kasinathan P, Sullivan E, Kuroiwa Y, Tomizuka K, Ishida I: Artificial chromosome vectors and expression of complex proteins in transgenic animals. Theriogenology. 2003, 59: 107-113.

    CAS  Article  PubMed  Google Scholar 

  55. Clark M: Antibody humanization: a case of the 'Emperor's new clothes'?. Immunol Today. 2000, 21: 397-402.

    CAS  Article  PubMed  Google Scholar 

  56. Boulianne GL, Hozumi N, Shulman MJ: Production of functional chimaeric mouse/human antibody. Nature. 1984, 312: 643-646.

    CAS  Article  PubMed  Google Scholar 

  57. Aya J: Production of fully human antibodies by transgenic mice. Current Opinion in Biotechnology. 1995, 6: 561-566.

    Article  Google Scholar 

  58. Lonberg N, Huszar D: Human antibodies from transgenic mice. Int Rev Immunol. 1995, 13: 65-93.

    CAS  Article  PubMed  Google Scholar 

  59. Mendez MJ, Green LL, Corvalan JR, Jia XC, Maynard-Currie CE, Yang XD, Gallo ML, Louie DM, Lee DV, Erickson KL, et al: Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice. Nat Genet. 1997, 15: 146-156.

    CAS  Article  PubMed  Google Scholar 

  60. Green LL, Hardy MC, Maynard-Currie CE, Tsuda H, Louie DM, Mendez MJ, Abderrahim H, Noguchi M, Smith DH, Zeng Y, et al: Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs. Nat Genet. 1994, 7: 13-21.

    CAS  Article  PubMed  Google Scholar 

  61. Kuroiwa Y, Kasinathan P, Choi YJ, Naeem R, Tomizuka K, Sullivan EJ, Knott JG, Duteau A, Goldsby RA, Osborne BA, et al: Cloned transchromosomic calves producing human immunoglobulin. Nat Biotechnol. 2002, 20: 889-894.

    CAS  Article  PubMed  Google Scholar 

  62. Grobet L, Martin LJ, Poncelet D, Pirottin D, Brouwers B, Riquet J, Schoeberlein A, Dunner S, Menissier F, Massabanda J, et al: A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nat Genet. 1997, 17: 71-74.

    CAS  Article  PubMed  Google Scholar 

  63. Elsea SH, Lucas RE: The mousetrap: what we can learn when the mouse model does not mimic the human disease. Ilar J. 2002, 43: 66-79.

    CAS  Article  PubMed  Google Scholar 

  64. Kent G, Iles R, Bear CE, Huan LJ, Griesenbach U, McKerlie C, Frndova H, Ackerley C, Gosselin D, Radzioch D, et al: Lung disease in mice with cystic fibrosis. J Clin Invest. 1997, 100: 3060-3069.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  65. Snouwaert JN, Brigman KK, Latour AM, Iraj E, Schwab U, Gilmour MI, Koller BH: A murine model of cystic fibrosis. Am J Respir Crit Care Med. 1995, 151: S59-64.

    CAS  Article  PubMed  Google Scholar 

  66. Harris A: Towards an ovine model of cystic fibrosis. Hum Mol Genet. 1997, 6: 2191-2194.

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank M. Julian for critical reading of this manuscript.

Author information

Affiliations

  1. Nexia Biotechnologies Inc., 1000, Ave. St-Charles, Vaudreuil Dorion, J7V 8P5, Quebec, Canada

    Bin Wang

  2. Neumain Inc., 170B, Ronald Drive, Montreal West, H4X 1M8, Quebec, Canada

    Jiangfeng Zhou

Authors
  1. Bin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jiangfeng Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bin Wang.

Electronic supplementary material

Authors’ original submitted files for images

Below are the links to the authors’ original submitted files for images.

Authors’ original file for figure 1

Authors’ original file for figure 2

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Wang, B., Zhou, J. Specific genetic modifications of domestic animals by gene targeting and animal cloning. Reprod Biol Endocrinol 1, 103 (2003). https://doi.org/10.1186/1477-7827-1-103

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/1477-7827-1-103

Keywords

  • Embryonic Stem Cell
  • Homologous Recombination
  • Prion Disease
  • Bovine Spongiform Encephalopathy
  • Scrapie

Reproductive Biology and Endocrinology

ISSN: 1477-7827

Contact us