Recent advances in the development of new transgenic animal technology

Abstract

Transgenic animal technology is one of the fastest growing biotechnology areas. It is used to integrate exogenous genes into the animal genome by genetic engineering technology so that these genes can be inherited and expressed by offspring. The transgenic efficiency and precise control of gene expression are the key limiting factors in the production of transgenic animals. A variety of transgenic technologies are available. Each has its own advantages and disadvantages and needs further study because of unresolved technical and safety issues. Further studies will allow transgenic technology to explore gene function, animal genetic improvement, bioreactors, animal disease models, and organ transplantation. This article reviews the recently developed animal transgenic technologies, including the germ line stem cell-mediated method to improve efficiency, gene targeting to improve accuracy, RNA interference-mediated gene silencing technology, zinc-finger nuclease gene targeting technology and induced pluripotent stem cell technology. These new transgenic techniques can provide a better platform to develop transgenic animals for breeding new animal varieties and promote the development of medical sciences, livestock production, and other fields.

Fig. 1

Generation of transgenic animals using spermatogonial stem cells. The testis from a fertile male is digested to generate a single-cell suspension. The SSCs can be cultured, transfected, and then microinjected into the lumen of seminiferous tubules of an infertile recipient mouse. Only a spermatogonial stem cell can generate a colony of spermatogenesis in the recipient testis. Mating the recipient male to a wild-type female produces progeny that carry foreign genes

Fig. 2

The three principal routes of ES cell, pronuclear injection, and somatic cell gene targeting technology to achieve targeted genome modifications in the rat and other mammalian species. ES cell embryonic stem cell, HR homologous recombination, ZFN zinc-finger nuclease, DSB double-strand break, NHEJ non-homologous end joining

Fig. 3

Diagrammatic representation for conditional gene targeting by Cre-loxp recombination. Two different strategies for production of knock-out and knock-in transgenic mice using Cre-loxp recombination system. The Cre-loxp system could be used to delete loxP-flanked DNA and to control site-specific recombination events in genomic DNA

Fig. 4

A schematic diagram of RNAi and miRNA pathways and strategies for RNAi-induced gene silencing in living mammals. Mammalian RNAi and miRNA pathways use different RNA substrates processed by the same Dicer protein into short RNAs. These processed RNAs are incorporated into the RNA-induced silencing complex (RISC), which targets messenger RNA to prevent translation. RNAi in animals can be induced from a transgene. Transgenic animals with RNAi knockdown are usually produced by one of the three depicted strategies (pronuclear injection, viral infection, and ESC route). Different strategies for delivering an RNAi agent are available to induce RNAi in adult animals

Fig. 5

Schematic depiction of production of mice using iPS cells and tetraploid complementation. Retrovirus-mediated transfection with four transcription factors (Oct4, Sox2, Klf4, and c-Myc) into mouse embryonic fibroblasts (MEF) has resulted in the generation of induced pluripotent stem (iPS) cells. Then iPS cells are injected into tetraploid blastocysts to generate iPSC mice