Clark Karl J, Carlson Daniel F, Foster Linda K, Kong Byung-Whi, Foster Douglas N, Fahrenkrug Scott C
Department of Animal Science, University of Minnesota, St. Paul, MN, USA.
BMC Biotechnol. 2007 Jul 17;7:42. doi: 10.1186/1472-6750-7-42.
Swine is an important agricultural commodity and biomedical model. Manipulation of the pig genome provides opportunity to improve production efficiency, enhance disease resistance, and add value to swine products. Genetic engineering can also expand the utility of pigs for modeling human disease, developing clinical treatment methodologies, or donating tissues for xenotransplantation. Realizing the full potential of pig genetic engineering requires translation of the complete repertoire of genetic tools currently employed in smaller model organisms to practical use in pigs.
Application of transposon and recombinase technologies for manipulation of the swine genome requires characterization of their activity in pig cells. We tested four transposon systems- Sleeping Beauty, Tol2, piggyBac, and Passport in cultured porcine cells. Transposons increased the efficiency of DNA integration up to 28-fold above background and provided for precise delivery of 1 to 15 transgenes per cell. Both Cre and Flp recombinase were functional in pig cells as measured by their ability to remove a positive-negative selection cassette from 16 independent clones and over 20 independent genomic locations. We also demonstrated a Cre-dependent genetic switch capable of eliminating an intervening positive-negative selection cassette and activating GFP expression from episomal and genome-resident transposons.
We have demonstrated for the first time that transposons and recombinases are capable of mobilizing DNA into and out of the porcine genome in a precise and efficient manner. This study provides the basis for developing transposon and recombinase based tools for genetic engineering of the swine genome.
猪是一种重要的农业商品和生物医学模型。对猪基因组进行操作可提供提高生产效率、增强抗病能力以及增加猪产品附加值的机会。基因工程还可以扩大猪在人类疾病建模、临床治疗方法开发或为异种移植提供组织方面的用途。要充分发挥猪基因工程的潜力，需要将目前在较小模式生物中使用的全套基因工具转化为在猪中的实际应用。
转座子和重组酶技术在猪基因组操作中的应用需要表征它们在猪细胞中的活性。我们在培养的猪细胞中测试了四种转座子系统——睡美人转座子、Tol2转座子、piggyBac转座子和护照转座子。转座子使DNA整合效率比背景提高了28倍，并能为每个细胞精确传递1至15个转基因。通过从16个独立克隆和20多个独立基因组位点去除正负选择盒的能力来衡量，Cre和Flp重组酶在猪细胞中均有功能。我们还展示了一种依赖Cre的基因开关，它能够消除中间的正负选择盒，并激活游离型和基因组驻留型转座子中的绿色荧光蛋白表达。
我们首次证明转座子和重组酶能够以精确有效的方式将DNA导入和导出猪基因组。本研究为开发基于转座子和重组酶的猪基因组基因工程工具奠定了基础。
