Future technology — marriage of nuclear transfer and advanced molecular tools

4. Future technology — marriage of nuclear transfer and advanced molecular tools

Ž Recent reports on the generation of transgenic sheep and cattle Schnieke et al., 1997; . Cibelli et al., 1998b via somatic nuclear transfer inspired great expectations about this elegant approach to improve the generation of transgenic livestock. Fetal fibroblasts Fig. 1. were transfected in vitro, screened for transgene integration and then transferred into enucleated oocytes. After fusion of both components and activation of the reconstituted nuclear transfer complexes, blastocysts were transfered to synchronized recipients and Ž . gave rise to transgenic offspring Fig. 1 . Compared with the microinjection procedure in which screening for transgenesis and optimal expression of the transgene takes place at the level of the offspring, cloning by nuclear transfer can accelerate the time-consum- ing transgenic production by prescreening of donor cells for the optimal expression of the desired trait in vitro and 100 transgenic offspring. As of today a variety of cell types has been successfully employed as donors in Ž . nuclear transfer Fulka et al., 1998 . However, the overall efficiency of nuclear transfer is low. Factors affecting the success of nuclear transfer are poorly defined and the percentage of live offspring does not exceed 1–3 of the transferred reconstituted Ž . embryos Cibelli et al., 1998b; Wilmut et al., 1997; Wakayama et al., 1998 . A better understanding of the underlying fundamental molecular processes, such as cell cycle Ž . compatibilities between cytoplasm and donor nucleus Campbell et al., 1996 , cell cycle Ž . synchronization of the donor cells Boquest et al., 1999; Kues et al., 2000 , reprogram- ming and the relevance of differentiation vs. totipotency is urgently needed. Upon serum deprivation or treatment with chemical cell cycle inhibitors, the majority of porcine donor cells was synchronized at the presumptive optimal cell cycle stage at G rG 1 Ž . without compromising their viability Kues et al., 2000 . This contributes substantially to standardize the nuclear transfer procedure as much as possible. In addition, methods have to be established that allow reliable determination of the capacity of a given nuclear transfer embryo to develop into a normal offspring. Currently, an increased peri- and postnatal mortality is found in offspring derived from nuclear transfer embryos Ž . Wilmut et al., 1997; Kato et al., 1998 . 4.1. Permanent cell lines ES, EG, EC The basis for loss of function transgenics in the mouse are the availability of Ž . embryonic stem cells ES cells , molecular tools for homologous recombination and the high probability after injection into host blastocysts with which ES cells give rise to Ž . germline contribution Evans and Kaufman, 1981; Martin, 1981 . This provides a powerful approach to introduce specific genetic changes into the murine genome. The essential characteristics of ES cells include derivation from the preimplantation embryo Ž . inner cell mass cells , undifferentiated proliferation in vitro and the developmental potential to differentiate into all cell types. Morphological markers for ES cells are the growth in three dimensional colonies, formation of embryoid bodies in vitro and of Ž . teratocarcinomas upon transplantation in immunodeficient mice Hogan et al., 1994 . Molecular markers used for murine ES cells are the stage-specific embryonic antigen-1 Ž . Ž . SSEA-1 , SSEA-3, SSEA-4, TA-60-1, octamer-binding transcription factor-4 Oct-4 , alcaline phosphatase activity, high telomerase activity as well as a lack of differentiation Ž . markers Hogan et al., 1994 . Ž . Ž . Besides ES cells, embryonic germ EG and embryonic carcinoma cells EC have been established in the mouse model. EG cells are isolated from cultured primordial Ž . Ž . germ cells PGC Matsui et al., 1992; Resnick et al., 1992 and share several characteristics with ES cells, including morphology, pluripotency, and the capacity for germline transmission. Similar to ES cells, EG cells express alkaline phosphatase and Oct-4. They can be aggregated to form embryoid bodies and give rise to teratocarcino- mas when transferred to appropriate ectopic sites. However, up to now, homologous recombination has not been achieved in this cell type. Murine EC cells were originally Ž . derived from induced teratocarcinomas Hogan et al., 1994 . However, they differentiate only rarely into gamete cells of reconstituted chimeras and are therefore not applicable for transgenic animal production. Thus, the ultimate criterion for true totipotent stem cell lines is the contribution to the germline either in chimeras or by starting a new development upon nuclear transfer. ES- Ž and EG-like cell lines have been isolated from sheep, pig and cattle Wheeler, 1994; . Anderson, 1999 . Porcine and bovine cell lines were capable of contributing to chimera Ž formation upon injection into appropriate host blastocysts Wheeler, 1994; Shim et al., . 1997; Cibelli et al., 1998a; Piedrahita el al., 1998 . However, no germline transmission has been reported so far. True totipotent stem cell lines might require specific culture conditions, growth factor supplements and probably a specific genetic background, as Ž . even only few mouse strains are suitable for ES cell isolation Hogan et al., 1994 . 4.2. Homologous recombination in liÕestock Homologous recombination in murine ES cells is the most direct and unambiguous way to eliminate gene function and is therefore the preferred method to establish a null genotype. Several strategies for gene targeting in murine ES cells have been developed Ž . Mayford et al., 1995; Kuhn and Schwenk, 1997; Muller, 1999 . More than 1000 ¨ ¨ Ž knockout strains have been created via gene targeting in ES cells mouse knockout and . mutation database www.biomednet.com . Prominent examples of gene knockouts are related to certain oncogenes, kinases, phosphatases, growth factors, transcription factors Ž and disease associated genes Bunz et al., 1998; Shastry, 1998; Gotz et al., 1998; Muller, ¨ ¨ . 1999 . The potential for a gene knockout technique in livestock production is high- lighted by the discovery that several beef cattle breeds, like Belgian Blue and Piedmon- tese, are accidentally homozygous for a mutated myostatin gene, which is functionally Ž inactive and could be referred to as a natural knockout Grobet et al., 1997; Kambadur et . al., 1997; McPherron and Lee, 1997 . The similarity in phenotypes of myostatin mutated Ž . cattle and myostatin null mice McPherron et al., 1997 is striking and suggests that myostatin is a potentially useful target for genetic modification in farm animals. Gene targeting in farm animals is hampered by the lack of true totipotent stem cells. However, the definition of totipotency has to be reconsidered, since the successful Ž cloning of sheep and cattle from adult mammary epithelial cells and fibroblasts Wilmut . et al., 1997; Cibelli et al., 1998b . Nuclear transfer techniques promise to circumvent the need for true totipotent cells for the generation of loss-of-function transgenic livestock. The future challenge in transgenic farm animal production is the isolation and handling Ž of primary cell cultures, either from somatic or embryonic origin Schnieke et al., 1997; . Cibelli et al., 1998b; Kues et al., 1998 . These could be used for sophisticated genetic modifications, clonal selection and subsequently for nuclear transfer. Gene targeting in somatic cells of livestock will have important applications in combination with nuclear transfer. Although gene targeting in somatic cells is relatively uncommon, recent Ž . Ž progress in rat cells Mateyak et al., 1997 and non-immortalized human cells Brown et . al., 1997; Bunz et al., 1998 clearly shows the significant potential of this field. Moreover, isogenic DNA does not seem to be a prerequisite for efficient gene targeting Ž . in human somatic cells Sedivy and Dutriaux, 1999 . Recently, Pharmaceutical Proteins Ž . Limited PPL has announced the birth of lambs produced by nuclear transfer of gene Ž targeted somatic cells, but no details have been disclosed Transgenic Animal Research . Conference, August 1999, Lake Tahoe, CA, USA . Gene targeting in somatic cells has been difficult to achieve because the absolute frequency of homologous recombination events in somatic cells is two orders of Ž magnitude lower than in ES cells Arbones et al., 1994; Hanson and Sedivy, 1995; . Ž . Brown et al., 1997 and the frequency of nonhomologous illegitimate recombination events is typically high. Targeting constructs must provide an efficient enrichment of homologously over nonhomologously recombined clones. The classical positive–nega- tive strategy uses vectors that are based on a negatively selectable gene which is placed on the flanks of the targeting sequences, and which is removed in the homologous recombination process. Additionally, an independent expression cassette for positive selection is needed. In contrast, in a promoterless targeting strategy the positively selectable marker lacks a suitable promoter and is driven by the endogenous promoter of the target gene after homologous recombination. Nonhomologous recombination events by random integration close to a chromosomal promoter seem to be relative rare Ž . Hanson and Sedivy, 1995 . These techniques promise to combine gene targeting in somatic cells and nuclear transfer for progress in transgenic large animal production. 4.3. Conditional mutagenesis and region specific-knockouts The above findings demonstrate the power of gene targeting, but they also point to a need for additional molecular techniques when precise genetic modifications are desired. Gene knockouts per se have no spatial or temporal restriction. As the targeted gene product is absent for the entire life of the animal in all cells, it is difficult to assign a phenotype to a specific knockout as a mutant organism may compensate for the loss of a gene product or the knockout may have complex, secondary effects, e.g. depending on Ž . the genetic background Gerlai, 1996 , or may even result in embryonic lethality. A powerful tool for the design of genetic switches and for accelerating the creation of genetically modified animals, is the Cre site-specific DNA recombinase of bacteriophage Ž . P1 Sauer, 1998 . A single 38-kDa Cre protein is required to catalyze recombination between two loxP recognition sites, consisting of 34-bp DNA sequences. Recombination can occur between directly repeated loxP sites on the same molecule to excise the intervening DNA sequence, irrespective of whether the recognition sites are located on a Ž . plasmid or a mammalian chromosome Sauer and Henderson, 1988 . The combination of tissue-specific promoter elements with the Cre DNA recombinase enables restriction Ž . of a gene knockout to a certain cell type or tissue Orban et al., 1992; Gu et al., 1994 . Two mouse strains are required for this approach. The first is a conventional transgenic strain in which the DNA recombinase is expressed in a cell-type-restricted manner. The second strain is generated by using gene targeting in ES cells. The second strain carries the target gene flanked on either end by loxP sequences. A crucial caveat is that the insertion of these loxP sites must not disrupt the normal function of the target gene. The two genetic alterations are brought together in one mouse through mating. This will give rise to a line of mice in which the target gene is deleted only in those cells that express the recombinase. Deletion of the DNA polymerase b gene in the germline resulted in a lethal phenotype, but deletion of this gene in T cells gave rise to viable animals in which Ž . the effects of the knockout in a specific cell type can be analyzed Gu et al., 1994 . Moreover, employing an inducible promoter, like the interferon-responsive promoter to control the expression of Cre recombinase allows insertion of an inducible gene Ž . knockout Kuhn et al., 1995 . In principle, recombinase-mediated recombination should ¨ also allow gene replacement, e.g. the exchange of the reading frames of milk protein genes with the sequences of genes encoding pharmaceutical proteins.

5. Conclusions and outlook