Pronuclear DNA microinjectionThe fi

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 controversially after its first description in the mouse, SMGT in the pig was achieved by collection of sperm, incubation of sperm with exogenous DNA, and artificial insemination of gilts with DNA-loaded sperm. An important factor for the success of this method seems to be the selection of suitable sperm donor animals [16]. Linker-based sperm-mediated gene transfer is a variant of the procedure where the uptake of exogenous DNA by sperm cells is improved by receptormediated endocytosis of DNA–antibody complexes [17]. Another modification of SMGT is intracytoplasmic sperm injection-mediated gene transfer. The first step is the induction of sperm membrane damage (e.g., by freezethawing), followed by incubation with exogenous DNA, and finally intracytoplasmic injection of sperm with bound DNA into oocytes [18].
Lentiviral gene transfer
Lentiviruses belong to the family Retroviridae and transfer their RNA genome into infected cells, where it is reverse transcribed to DNA and integrated into the host genome as a so-called provirus which is transmitted in Mendelian manner to the offspring. Lentiviruses can transduce nondividing cells which allows immediate integration of the vector genome into the early embryo, reducing the risk of mosaic formation [19]. Lentiviral gene transfer was adapted to pigs [20, 21] and resulted in high proportions of transgenic offspring. Although lentiviral vector systems can only carry

Kết quả (Việt) 1:

Pronuclear DNA microinjectionCác kỹ thuật đầu tiên thành công được sử dụng để sản xuất biến đổi gen con lợn là DNA microinjection vào pronuclei của zygotes [13, 14]. Nói chung, hiệu quả của DNA microinjection là thấp. Ngoài ra, pronuclear DNA microinjection bị từ thực tế là nó có thể mang lại động vật sáng lập654 J Mol Med (2010) 88:653-664Mosaic, và rằng các tích hợp ngẫu nhiên của DNA tiêm mảnh có thể gây ra mức độ biểu hiện khác nhau do vị trí tác động của DNA giáp ranh hoặc có thể phá vỡ chức năng nội sinh chuỗi (insertional mutagenesis; xem xét trong [4]). Mặc dù hiệu quả nói chung thấp, có lẽ hầu hết các dòng biến đổi gen lợn sẵn có cho đến nay đã được thành lập bởi kỹ thuật pronuclear microinjection.Gen tinh trùng trung gian chuyển giaoSMGT dựa trên khả năng nội tại của tinh trùng để ràng buộc và trong lòng ngoại sinh DNA và chuyển nó vào trứng trong thụ tinh (xem xét trong [15]). Mặc dù hiệu quả của SMGT đã được thảo luận controversially sau khi mô tả đầu tiên của nó trong chuột, SMGT trong con lợn đạt được bằng bộ sưu tập của tinh trùng, ủ bệnh của tinh trùng với ADN ngoại sinh, và thụ tinh nhân tạo của gilts với DNA-nạp tinh trùng. Một yếu tố quan trọng cho sự thành công của phương pháp này có vẻ là lựa chọn của động vật nhà tài trợ tinh trùng phù hợp [16]. Dựa trên Linker trung gian tinh trùng gen chuyển là một biến thể của các thủ tục mà sự hấp thu của DNA ngoại sinh bởi các tế bào tinh trùng được cải thiện bởi receptormediated endocytosis của tổ hợp DNA-kháng thể [17]. Một sửa đổi của SMGT là tinh trùng intracytoplasmic tiêm trung gian gen chuyển. Bước đầu tiên là cảm ứng của thiệt hại màng tế bào tinh trùng (ví dụ:, bởi freezethawing), theo sau là ấp trứng với DNA ngoại sinh, và cuối cùng intracytoplasmic tiêm tinh trùng với ADN bị ràng buộc vào oocytes [18].Lentiviral gen chuyểnLentiviruses belong to the family Retroviridae and transfer their RNA genome into infected cells, where it is reverse transcribed to DNA and integrated into the host genome as a so-called provirus which is transmitted in Mendelian manner to the offspring. Lentiviruses can transduce nondividing cells which allows immediate integration of the vector genome into the early embryo, reducing the risk of mosaic formation [19]. Lentiviral gene transfer was adapted to pigs [20, 21] and resulted in high proportions of transgenic offspring. Although lentiviral vector systems can only carry <10 kb exogenous DNA, this is considered to be enough for transfer of expression vectors for cDNAs and small interfering RNAs. As prokaryotic vector sequences are often subject to epigenetic silencing by DNA methylation, it was important to investigate this phenomenon in transgenic pigs harboring lentiviral integrants. Our studies revealed that—after segregation to the G1 generation—one third of lentiviral integrants exhibited low expression levels and hypermethylation [22], whereas two thirds of the lentiviral integrants were expressed faithfully through subsequent generations. Thus, lentiviral transgenesis is clearly an attractive alternative to the pronuclear microinjection technique.Somatic cell nuclear transferSince successful SCNT protocols are available for the pig [23–25], this technology is an attractive route for geneticHình 1 kỹ thuật hiện tại để sửa đổi di truyền của con lợn bao gồm DNA microinjection vào pronuclei của thụ tinh oocytes (DNA-MI), chuyển giao cho gen spermmediated (SMGT), lentiviral transgenesis (LV-GT), và tế bào Soma hạt nhân chuyển sử dụng biến đổi nhà tài trợ hạt nhân tế bào (SCNT). LV-GT có thể được thực hiện bởi subzonal tiêm của virus hạt vào oocytes trước hoặc sau khi thụ tinh. Một biến thể của SMGT là intracytoplasmic tiêm (ICSI) của tinh trùng đông lạnh-xả đá sau khi ủ bệnh ADN (xem các văn bản cho biết thêm chi tiết)J Mol Med (2010) 88:653-664 655modification of this species. In general, transgenesis by SCNT involves the following steps: (1) genetic modification and selection of donor cells in culture; (2) recovery and enucleation of in vivo or in vitro matured oocytes (metaphase II); (3) nuclear transfer by electrofusion or piezo-actuated microinjection (less common) and activation; (4) in vitro culture of the reconstructed embryos; and (5) embryo transfer to synchronized recipients. Various modifications like “handmade cloning” were developed to simplify SCNT in pigs [26]. SCNT is so far the only route to introduce targeted mutations into the pig genome. Via homologous recombination in nuclear donor cells, mutations have been introduced in the alpha-1,3-galactosyltransferase (GGTA1) and the cystic fibrosis transmembrane conductance regulator (CFTR) genes, and live offspring have been born following SCNT using these cells [27, 28]. Other attractive characteristics comprise: no generation of mosaic phenotypes and the possibility of pre-selection of donor cells with regard to transgene expression or gender. Furthermore, SCNT from genetically modified pools of donor cells followed by selection of suitable donor fetuses or offspring can be used to speed up transgenesis in the pig (Fig. 2). The efficiency of cloning in pig is still relatively low, ranging between 0.5% and 5% offspring per transferred SCNT embryos. As in other species the low efficiency of SCNT is attributed to failures in epigenetic reprogramming (reviewed in [29]).Transgenic pigs as models for human diseasesCompared to laboratory rodents, experimental standardization of large animal models is low, and cost and labor are high. Therefore, transgenic pig models have been primarily developed for important disease areas where translational research in the available rodent models is limited by their small size and short life span or where rodent models do not adequately reflect the respective disease phenotypes. A summary of published transgenic pig models is provided in Table 1. Referring to the genes described in Table 1, protein data of amyloid precursor protein (APP), huntingtin (HTT), rhodopsin (RHO), endothelial cell nitric oxide synthase (eNOS), CFTR, and hepatocyte nuclear factor 1 alpha (HNF1A) are available for human, pig, and mouse (http:// www.uniprot.org; http://www.ensembl.org). Species comparison of orthologous proteins was done by alignment and manual adaptation of the amino acid sequences in BioEdit [30]. For APP, RHO, eNOS, CFTR, and HNF1A, the human proteins are more similar to the pig orthologs. For HTT, the human protein is more similar to the mouse ortholog. However, analysis of the repeat number of intragenic trinucleotide repeats associated with inheritedhuman neurodegenerative diseases showed that the trinucleotide repeat regions are more conserved in terms of repeat length between humans and pigs than between humans and rodents [31].Neurodegenerative diseasesAlzheimer’s disease is a multifactorial neural disease and occurs in some families as an autosomal dominant disorder showing the onset of the disease after 40 years. Causative mutations were identified in the amyloid precursor protein gene (APP) leading to the increased production of distinct protein fragments which in turn results in neuropathy. To develop a pig model for Alzheimer’s disease, transgenic pigs were produced using the human dominant mutant allele APPsw harboring two amino acid exchanges due to two neighboring nucleotide exchanges which was found to cause Alzheimer’s disease. According to previous transgenic mouse studies, a 7.5 kb transgene was constructed with a 1-kb platelet-derived growth factor-beta promoter, intronic and exonic sequences of the beta-globin gene, the cDNA encoding the mutant allele APPsw, and SV40 polyadenylation sequences. After stable genetic modification of fibroblasts of the Göttingen minipig breed, one transgenic cell clone was used for SCNT to produce seven healthy transgenic cloned pigs with normal weight gain. The transgenic pigs harbored a single full-length copy of the transgene in their genome and showed strong, promoterspecific expression of the transgenic protein in brain tissues. Accumulation of the pathogenic protein and subsequent appearance of clinical consequences were estimated to develop with increasing age [32]. Huntington’s disease is an autosomal dominant, progressive neurodegenerative disorder involving the premature loss of specific neurons. It is associated with an expansion of a CAG trinucleotide repeat in the 5′ region of the huntingtin gene (HTT) which results in a lengthened polyglutamine tract of the protein. The CAG repeat number is polymorphic, ranging from 6 to 35 units in normal alleles and from 36 to 120 units in alleles associated with Huntington’s disease. The 12.8-kb HTT transcript of Göttingen minipigs codes for a 345-kDa protein (3,139 amino acids) [33]. The 8.2-kb transgene used for DNA microinjection into minipig embryos consisted of the 4-kb rat neuronspecific enolase (Nse) promoter, a 3.3-kb 5 ′ minipig huntingtin cDNA which was mutated by insertion of 75 CAG repeats into the triplet region of exon 1, and a 0.9-kb SV40 polyadenylation signal. Five transgenic founder pigs were produced each harboring one to three different integration sites with variable copy numbers and indication of genetic mosaicism [34]. To date, no follow-up publication describing mutant phenotypes appeared.656 J Mol Med (2010) 88:653–664Retinitis pigmentosa (RP) typically causes night blindness early in life due to loss of rod photoreceptors. The remaining cone photoreceptors slowly degenerate leading ultimately to blindness. Various genes and loci are associated with thedisease. Both transgenic and knockout rodent models of retinal dystrophy contributed to the analysis of the disease. Compared to humans, rodent models are limited by two disadvantages, the small number and different distribution ofneopromoter coding sequence Expressing transgenic pigsSelectable expression vectorTransfectionSelection Embryo transferNuclear transfer INuclear transfer IIEstablishment