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TALE nickase-mediated SP110 knockin endows cattle with increased resistance to tuberculosis
Friday, 2015/04/03 | 08:06:21

Haibo Wu, Yongsheng Wang, Yan Zhang, Mingqi Yang, Jiaxing Lv, Jun Liu, and Yong Zhang

Significance

Bovine tuberculosis is a chronic infectious disease that affects a broad range of mammalian hosts. It is a serious threat to agriculture in many less-developed countries. In this study, we introduced a mutation to the FokI of the right hand of wild-type transcription activator-like effector nuclease and established a transcription activator-like effector nickase system that creates single-strand breaks in the genome. Then we used this system to add the mouse gene SP110 to a specific location in the bovine genome and created transgenic cattle with increased resistance to tuberculosis. Our results contribute to the control and prevention of bovine tuberculosis and provide a previously unidentified insight into breeding animals for disease resistance.

Abstract

Transcription activator-like effector nuclease (TALEN)-mediated genome modification has been applied successfully to create transgenic animals in various species, such as mouse, pig, and even monkey. However, transgenic cattle with gene knockin have yet to be created using TALENs. Here, we report site-specific knockin of the transcription activator-like effector (TALE) nickase-mediated SP110 nuclear body protein gene (SP110) via homologous recombination to produce tuberculosis-resistant cattle. In vitro and in vivo challenge and transmission experiments proved that the transgenic cattle are able to control the growth and multiplication of Mycobacterium bovis, turn on the apoptotic pathway of cell death instead of necrosis after infection, and efficiently resist the low dose of M. bovis transmitted from tuberculous cattle in nature. In this study, we developed TALE nickases to modify the genome of Holstein–Friesian cattle, thereby engineering a heritable genome modification that facilitates resistance to tuberculosis.

 

See http://www.pnas.org/content/112/13/E1530.abstract

PNAS March 31, 2015; vol.112 no. 13 E1530-E1539

Fig. 1.

Activity assessment of TALENs. (A) Schematic representation of the targeting locus. (B) Schematic representation of the TALEN constructs. The lengths of constructs were calculated based on TALEN-encoding plasmids with 17 modules. (C) Cleavage activity of each TALEN was measured by luciferase SSA assay in human 293-FT cells. Data are presented as mean ± SD and are derived from three independent experiments. **P < 0.01. (D) Frequency of allelic mutation as determined by Surveyor nuclease assays. Different amounts of each TALEN transfected are shown as indicated. (E) The M-S locus was PCR amplified, and cleavage of the locus was measured using a Surveyor nuclease assay. The degree of cleavage was quantified and is shown below each lane. (F) Some of the representative sequences revealed distinct TALEN-induced insertions and deletions at the targeted locus. The binding site of TALEN is underlined in red. Occurrences of deletions and insertions are listed on the right. The lowercase letters represent inserted bases

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