REVIEWTransgenic pigs as models for

REVIEW
Transgenic pigs as models for translational biomedical research
Bernhard Aigner & Simone Renner & Barbara Kessler & Nikolai Klymiuk & Mayuko Kurome & Annegret Wünsch & Eckhard Wolf
Received: 1 September 2009 /Revised: 26 February 2010 /Accepted: 2 March 2010 /Published online: 26 March 2010 # Springer-Verlag 2010
Abstract The translation of novel discoveries from basic research to clinical application is a long, often inefficient, and thus costly process. Accordingly, the process of drug development requires optimization both for economic and for ethical reasons, in order to provide patients with appropriate treatments in a reasonable time frame. Consequently, “Translational Medicine” became a top priority in national and international roadmaps of human health research. Appropriate animal models for the evaluation of efficacy and safety of new drugs or therapeutic concepts are critical for the success of translational research. In this context rodent models are most widely used. At present, transgenic pigs are increasingly being established as large animal models for selected human diseases. The first pig whole genome sequence and many other genomic resources will be available in the near future. Importantly, efficient and precise techniques for the genetic modification of pigs have been established, facilitating the generation of tailored disease models. This article provides an overview of the current techniques for genetic modification of pigs and the transgenic pig models established for neurodegenerative diseases, cardiovascular diseases, cystic fibrosis, and diabetes mellitus.
Keywords Pig.Genetic engineering.Animal model. Translational medicine
Introduction
The term “Translational Medicine” is being increasingly used to describe strategies of developing discoveries in basic research into clinically applicable novel therapies [1]. Despite increased efforts and investments into research and development, the output of novel pharmaceuticals has declined dramatically over the past years. The phenomenon of a retarded entry of new drugs and diagnostics to the market in spite of increased scientific discoveries and major financial investments is often addressed as “pipeline problem” [2]. This is attributed to the fact that currently used in vitro models, animal models, and early human trials do not reflect the patient situation well enough to reliably predict efficacy and safety of a novel compound or device. Advanced insights into the molecular pathogenesis of diseases lead to a plethora of innovative therapeutic concepts which address defined molecular targets. However, the translation of these concepts into clinical application requires a serial and systematic evaluation of efficacy and safety all the way through from discovery, preclinical science to the phases of clinical testing. The “Critical Path Initiative” of the US Food and Drug Administration (http://www.fda.gov/oc/initiatives/critical path/) focuses on the scientific developments that are necessary to realize the required systematic processes and mechanisms of evaluation. One of the leading topics is “Biomarker Development”, since biomarkers play major roles both in early (e.g., testing of efficacy and safety in animal models) and late phases of drug development (e.g., establishment of dose–response profiles, evaluation of sideeffects). Therefore, biomarker discovery and validation are also central themes in the “Innovative Medicine Initiative (IMI)” of the European Union (http://www.imi-europe.org/).
B. Aigner:S. Renner:B. Kessler:N. Klymiuk:M. Kurome: A. Wünsch:E. Wolf (*) Chair for Molecular Animal Breeding and Biotechnology, Department of Veterinary Sciences; and Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Feodor-Lynen-Str. 25, 81377 Munich, Germany e-mail: [e-mail]
J Mol Med (2010) 88:653–664 DOI 10.1007/s00109-010-0610-9
Biomarkers are objective and quantitative parameters that may serve as indicators of physiological processes, pathological changes as well as reactions to therapeutic intervention. The development of qualified biomarkers requires an integrated network of technology platforms. State-of-the-art technologies for molecular profiling at various levels (genome, transcriptome, proteome, metabolome, etc.) are connected with advanced techniques of bioimaging. Quantitative data from the different levels of information are integrated using the fast growing tools of bioinformatics and quantitative biology to optimize the predictionofefficacyandsafetyofnewdrugsandbiomarkers. Suitable animal models play a pivotal role in this process. Rodentmodelsare mostwidelyuseddue tothe possibility for genetic and environmental standardization, a broad spectrum of strains tailored to specific scientific problems, and their acceptancebytheregulatoryauthorities.Atpresent,transgenic pigs are increasingly established as additional large animal models for selected human diseases.
Pigs as models for translational research
Livestockpigbreedsandminiaturepigsarerelevantmodelsin many fields of medical research [3]. The omnivores human and pig have a large number of similarities in anatomy, physiology, metabolism, and pathology, e.g., they have a very similar gastrointestinal anatomy and function, pancreas morphology, and metabolic regulation. Moreover, pigs as large animal models are highly reproductive displaying early sexual maturity (with 5–8 months), a short generation interval (of 12 months), parturition of multiple offspring (an average of 10–12 piglets per litter), and all season breeding [4]. Standardization of the environment, i.e., pig housing,feeding,andhygienemanagement,iswelldeveloped [5]. Reproductive technology and techniques of genetic modification have considerably advanced in the last years (see below). Intense breeding efforts have provided pig breeds differing substantially in important traits such as size, metabolic characteristics, and behavior. If livestock pig breeds are employed for experimentation, the genetic background is mostly not defined. In contrast, minipig outbred stocks with full pedigree are delivered from commercial suppliers (http://www.minipigs.com/). In addition, inbred minipigs are available [6, 7]. Some pig breeds such as the Göttingen minipig® are used as non-rodent models for pharmacological and toxicological studies and are fully accepted by regulatory authorities worldwide (http:// www.minipigs.com/). As a member of the artiodactyls (cloven-hoofed mammals), the pig is evolutionarily distinct from the primates and rodents [8]. An initial evolutionary analysis based on
∼3.84 million shotgun sequences (0.66× coverage of the pig genome) and the available human and mouse genome data revealed that for each of the types of orthologous sequences investigated (e.g., exonic, intronic, intergenic, 5′ UTR, 3′ UTR, and miRNA), the pig is closer to human than mouse [9]. This was confirmed by the comparative analysis of protein coding sequences using full-length cDNA alignments comprising more than 700 kb from human, mouse, and pig where most gene trees favored a topology with rodents as outgroup to primates and artiodactyls [10]. A draft sequence of the whole pig genome is expected to be completed in the near future. The sequence data are being released through Ensembl (http://www.ensembl.org/Sus_ scrofa/Info/Index) as sequencing progresses. In addition to the genome-sequencing project, efforts were made in several groups to identify single nucleotide polymorphisms (SNP) through a substantial amount of shallow sequencing of additional breeds, resulting in a high-density (60 k) SNP chip distributed by Illumina, Inc. [11]. Recently, the so far largest collection of more than one million porcine-expressed sequence tags (ESTs) from 35 different tissues and three developmental stages was analyzed. This EST collection represents an essential resource for annotation, comparative genomics, assembly of the pig genome sequence, and further porcine transcriptome studies [12].
Genetic engineering of pigs
Importantly, pigs can be genetically modified to recapitulate the genetic and/or functional basis of a particular human disease, resulting in refined and tailored animal models for translational biomedical research. Current techniques for the genetic modification of pigs include DNA microinjection into the pronuclei of fertilized oocytes (DNA-MI), spermmediated gene transfer (SMGT), lentiviral transgenesis (LVGT), and somatic cell nuclear transfer using genetically modified nuclear donor cells (SCNT; Fig. 1). Other large non-primate animal models for human diseases include dogs and rabbits. Reproductive as well as transgenic techniques are poorly developed for dogs. Transgenic rabbits were produced using additive gene transfer, but no targeted mutations were introduced in the rabbit genome to date. In addition, the rabbit genome is sequenced only to a low coverage (http://www.ensembl.org).
Pronuclear DNA microinjection
The first technique successfully used to produce transgenic pigs was DNA microinjection into pronuclei of zygotes [13, 14]. Generally, the efficiency of DNA microinjection is low. In addition, pronuclear DNA microinjection suffers from the fact that it may yield founder animals that are
654 J Mol Med (2010) 88:653–664
mosaic, and that random integration of the injected DNA fragments may cause varying expression levels due to position effects of the neighboring DNA or may disrupt functional endogenous sequences (insertional mutagenesis; reviewed in [4]). In spite of the overall low efficiency, probably most of the transgenic pig lines existing so far have been established by the pronuclear microinjection technique.
Sperm-mediated gene transfer
SMGT is based on the intrinsic ability of sperm to bind and internalize exogenous DNA and to transfer it into the egg during fertilization (reviewed in [15]). Although the efficiency of SMGT was discussed controve

Kết quả (Việt) 1:

ĐÁNH GIÁBiến đổi gen con lợn là mô hình cho nghiên cứu y sinh tịnhBernhard Seehofer & Simone Renner & Barbara Kessler & Nikolai Klymiuk & Mayuko Kurome Annegret Wünsch & Eckhard WolfNhận được: 1 tháng 9 năm 2009/sửa đổi: tháng 2 năm 2010/chấp nhận: 2 tháng ba 2010 /Published trực tuyến: 26 Tháng ba năm 2010 # Springer-Verlag 2010Tóm tắt các bản dịch của tiểu thuyết phát hiện từ nghiên cứu cơ bản để ứng dụng lâm sàng là một quá trình dài, thường không hiệu quả, và do đó tốn kém. Theo đó, quá trình phát triển thuốc yêu cầu tối ưu hóa cả hai cho kinh tế và vì lý do đạo Đức, để cung cấp cho bệnh nhân với phương pháp điều trị thích hợp trong một khung thời gian hợp lý. Do đó, "Translational y học" đã trở thành một ưu tiên hàng đầu trong nước và quốc tế roadmaps nghiên cứu sức khỏe con người. Phù hợp các mô hình động vật để đánh giá hiệu quả và an toàn của loại thuốc mới hoặc điều trị khái niệm rất quan trọng cho sự thành công của nghiên cứu translational. Trong bối cảnh này động vật gặm nhấm mô hình được sử dụng rộng rãi nhất. Hiện nay, biến đổi gen con lợn đang ngày càng được thành lập như là mô hình động vật lớn đối với bệnh nhân đã chọn. Trình tự đầu tiên toàn bộ bộ gen con lợn và nhiều tài nguyên gen khác sẽ có sẵn trong tương lai gần. Quan trọng, hiệu quả và chính xác để sửa đổi di truyền của con lợn kỹ thuật đã được thành lập, tạo điều kiện cho các thế hệ của mô hình bệnh phù hợp. Bài viết này cung cấp một tổng quan về các kỹ thuật hiện tại cho các sửa đổi di truyền của con lợn và các mô hình biến đổi gen con lợn thành lập cho bệnh neurodegenerative, bệnh tim mạch, xơ nang, và bệnh tiểu đường.Từ khóa kỹ thuật Pig.Genetic. Động vật mô hình. Translational y họcGiới thiệuThe term “Translational Medicine” is being increasingly used to describe strategies of developing discoveries in basic research into clinically applicable novel therapies [1]. Despite increased efforts and investments into research and development, the output of novel pharmaceuticals has declined dramatically over the past years. The phenomenon of a retarded entry of new drugs and diagnostics to the market in spite of increased scientific discoveries and major financial investments is often addressed as “pipeline problem” [2]. This is attributed to the fact that currently used in vitro models, animal models, and early human trials do not reflect the patient situation well enough to reliably predict efficacy and safety of a novel compound or device. Advanced insights into the molecular pathogenesis of diseases lead to a plethora of innovative therapeutic concepts which address defined molecular targets. However, the translation of these concepts into clinical application requires a serial and systematic evaluation of efficacy and safety all the way through from discovery, preclinical science to the phases of clinical testing. The “Critical Path Initiative” of the US Food and Drug Administration (http://www.fda.gov/oc/initiatives/critical path/) focuses on the scientific developments that are necessary to realize the required systematic processes and mechanisms of evaluation. One of the leading topics is “Biomarker Development”, since biomarkers play major roles both in early (e.g., testing of efficacy and safety in animal models) and late phases of drug development (e.g., establishment of dose–response profiles, evaluation of sideeffects). Therefore, biomarker discovery and validation are also central themes in the “Innovative Medicine Initiative (IMI)” of the European Union (http://www.imi-europe.org/).B. Aigner:S. Renner:B. Kessler:N. Klymiuk:M. Kurome: A. Wünsch:E. Wolf (*) Chair for Molecular Animal Breeding and Biotechnology, Department of Veterinary Sciences; and Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Feodor-Lynen-Str. 25, 81377 Munich, Germany e-mail: [e-mail] Mol Med (2010) 88:653–664 DOI 10.1007/s00109-010-0610-9Biomarkers are objective and quantitative parameters that may serve as indicators of physiological processes, pathological changes as well as reactions to therapeutic intervention. The development of qualified biomarkers requires an integrated network of technology platforms. State-of-the-art technologies for molecular profiling at various levels (genome, transcriptome, proteome, metabolome, etc.) are connected with advanced techniques of bioimaging. Quantitative data from the different levels of information are integrated using the fast growing tools of bioinformatics and quantitative biology to optimize the predictionofefficacyandsafetyofnewdrugsandbiomarkers. Suitable animal models play a pivotal role in this process. Rodentmodelsare mostwidelyuseddue tothe possibility for genetic and environmental standardization, a broad spectrum of strains tailored to specific scientific problems, and their acceptancebytheregulatoryauthorities.Atpresent,transgenic pigs are increasingly established as additional large animal models for selected human diseases.Pigs as models for translational researchLivestockpigbreedsandminiaturepigsarerelevantmodelsin many fields of medical research [3]. The omnivores human and pig have a large number of similarities in anatomy, physiology, metabolism, and pathology, e.g., they have a very similar gastrointestinal anatomy and function, pancreas morphology, and metabolic regulation. Moreover, pigs as large animal models are highly reproductive displaying early sexual maturity (with 5–8 months), a short generation interval (of 12 months), parturition of multiple offspring (an average of 10–12 piglets per litter), and all season breeding [4]. Standardization of the environment, i.e., pig housing,feeding,andhygienemanagement,iswelldeveloped [5]. Reproductive technology and techniques of genetic modification have considerably advanced in the last years (see below). Intense breeding efforts have provided pig breeds differing substantially in important traits such as size, metabolic characteristics, and behavior. If livestock pig breeds are employed for experimentation, the genetic background is mostly not defined. In contrast, minipig outbred stocks with full pedigree are delivered from commercial suppliers (http://www.minipigs.com/). In addition, inbred minipigs are available [6, 7]. Some pig breeds such as the Göttingen minipig® are used as non-rodent models for pharmacological and toxicological studies and are fully accepted by regulatory authorities worldwide (http:// www.minipigs.com/). As a member of the artiodactyls (cloven-hoofed mammals), the pig is evolutionarily distinct from the primates and rodents [8]. An initial evolutionary analysis based on∼3.84 million shotgun sequences (0.66× coverage of the pig genome) and the available human and mouse genome data revealed that for each of the types of orthologous sequences investigated (e.g., exonic, intronic, intergenic, 5′ UTR, 3′ UTR, and miRNA), the pig is closer to human than mouse [9]. This was confirmed by the comparative analysis of protein coding sequences using full-length cDNA alignments comprising more than 700 kb from human, mouse, and pig where most gene trees favored a topology with rodents as outgroup to primates and artiodactyls [10]. A draft sequence of the whole pig genome is expected to be completed in the near future. The sequence data are being released through Ensembl (http://www.ensembl.org/Sus_ scrofa/Info/Index) as sequencing progresses. In addition to the genome-sequencing project, efforts were made in several groups to identify single nucleotide polymorphisms (SNP) through a substantial amount of shallow sequencing of additional breeds, resulting in a high-density (60 k) SNP chip distributed by Illumina, Inc. [11]. Recently, the so far largest collection of more than one million porcine-expressed sequence tags (ESTs) from 35 different tissues and three developmental stages was analyzed. This EST collection represents an essential resource for annotation, comparative genomics, assembly of the pig genome sequence, and further porcine transcriptome studies [12].Genetic engineering of pigs
Importantly, pigs can be genetically modified to recapitulate the genetic and/or functional basis of a particular human disease, resulting in refined and tailored animal models for translational biomedical research. Current techniques for the genetic modification of pigs include DNA microinjection into the pronuclei of fertilized oocytes (DNA-MI), spermmediated gene transfer (SMGT), lentiviral transgenesis (LVGT), and somatic cell nuclear transfer using genetically modified nuclear donor cells (SCNT; Fig. 1). Other large non-primate animal models for human diseases include dogs and rabbits. Reproductive as well as transgenic techniques are poorly developed for dogs. Transgenic rabbits were produced using additive gene transfer, but no targeted mutations were introduced in the rabbit genome to date. In addition, the rabbit genome is sequenced only to a low coverage (http://www.ensembl.org).
Pronuclear DNA microinjection
The first technique successfully used to produce transgenic pigs was DNA microinjection into pronuclei of zygotes [13, 14]. Generally, the efficiency of DNA microinjection is low. In addition, pronuclear DNA microinjection suffers from the fact that it may yield founder animals that are
654 J Mol Med (2010) 88:653–664
mosaic, and that random integration of the injected DNA fragments may cause varying expression levels due to position effects of the neighboring DNA or may disrupt functional endogenous sequences (insertional mutagenesis; reviewed in [4]). In spite of the overall low efficiency, probably most of the transgenic pig lines existing so far have been established by the pronuclear microinjection technique.
Sperm-mediated gene transfer
SMGT is based on the intrinsic ability of sperm to bind and internalize exogenous DNA and to transfer it into the egg during fertilization (reviewed in [15]). Although the efficiency of SMGT was discussed controve