TALE nickase-mediated SP110 knockin endows cattle with increased resistance to tuberculosis

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.

Keywords: TALEN; disease resistance; homologous recombination; single-strand break; tuberculosis.

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.

Fig. 2.

TALE nickase induced an SSB at the M-S locus and eliminated the NHEJ repair pathway. (A) Illustration of the expected digestion patterns following TALE nickase cleavage when resolved under nondenaturing and denaturing conditions. (B) Actual results of SSB when resolved under nondenaturing and denaturing conditions. (C) Cleavage activity of TALE nickase was measured by Surveyor nuclease assay. TALE nickase decreased DNA cleavage activity, but no NHEJ events were detected.

Fig. 3.

Targeted and heritable addition of the SP110 gene using TALE nickase. (A) Schematic representation of the gene-targeting vector. (B) Schematic overview depicting the targeting strategy for SP110. D450A, FokI bearing a D450A mutation. (C) Schematic overview screening the individual colonies. 5j F, lr F, and 3j R, lr R are primers for regions outside the homologous arms; 5j R and 3j F are primers for the targeting vector region. Southern blot probes are shown as red lines; Hind III digestion is used in Southern blot analysis. (D and E) Southern blot analysis of the nine heterozygous donor cells used for SCNT. (D) A 6.8-kb band resulting from targeted inclusion of the SP110 cassette was detected in addition to the 5.9-kb wild-type band when probe 1 was used. (E) Only a 6.8-kb targeted band was detected when probe 2 was used.

Fig. 4.

Assessment of transgenic cattle. (A) Photographs of SP110 gene-targeted calves that lived longer than 6 mo. The legends in the photographs identify the origin of donor cell lines. (B–D) 5′-junction (B, 1.49 kb), 3′-junction (C, 1.67 kb), and long-range (D, wild type: 1.64 kb; targeted: 5.98 kb) PCR to confirm site-specific targeting in transgenic cattle. Templates for PCR were genomic DNA extracted from cattle peripheral blood. Con, control normal cattle. Lanes 1–13 represent the 13 live calves. Lanes 14–17 represent four randomly selected dead transgenic calves. (E) Nucleotide sequence between endogenous and exogenous DNA corresponding to homologous recombination in transgenic cattle. (F and G) Southern blot analysis of the genomic DNA extracted from transgenic cattle. Lanes 1–13 represent the 13 live calves. Lanes 14–17 represent four randomly selected dead transgenic calves.

Fig. 5.

Assessment of the ability of transgenic cattle to resist tuberculosis. (A) SP110 was expressed correctly in macrophages isolated from transgenic cattle. Lanes 1–13 represent the 13 live transgenic cattle. Con, control cattle. (B) SP110 was expressed only in macrophages. Organs were obtained from a pool of dead transgenic cattle. Milk was obtained from three live transgenic cattle. Con, control donor cells; MP, macrophages. (C) The addition of SP110 did not affect the expression of nearby endogenous genes. Macrophages were separated from transgenic cattle (n = 9) or control cattle (n = 9). The relative expression levels of SFTPD, MBL1, SFTPA1, and MAT1A were detected by real-time RT-PCR. Each sample was tested individually, but data were analyzed by group. (D) Multiplication of M. bovis in macrophages from control (n = 9) or transgenic (n = 9) cattle in vitro. The macrophages were separated from each animal individually and were mixed by group. M. bovis multiplication was determined by cfu assays. (E) Flow cytometry analysis of the mechanism of cell death of the transgenic cattle macrophages infected with M. bovis. Early apoptotic [annexin V⁺ propidium iodide (PI)⁻] late apoptotic (annexin V⁺ PI⁺), and necrotic (annexin V⁻ PI⁺). (Left) Normal macrophages. (Middle) Infected experiment control macrophages. (Right) Infected transgenic macrophages. (F) M. bovis bacterial loads in the organs of the transgenic cattle after endobronchial infection. (G) Amount of IFN-γ produced in experimental control (n = 9) and transgenic (n = 9) cattle that shared a confined airspace with positive control cattle for 12 wk. (H) Concentrations of ESAT-6 and CFP-10 IFN-γ–producing SFCs in PBMCs of control and transgenic cattle. (I) H&E stains show a tubercle in the hilar lymph node of the control cattle (A and C) and normal tissue of transgenic cattle (B and D) 16 wk after infection. Arrows show the Langhans giant cells in the tubercle. (Magnification: 100× in I, a and b; 400× in I, c and d.) (Scale bars: 50 μm.) The transgenic cattle were divided into three groups according to their origin (derived from three different BFFs), and three cattle were picked randomly from each group for the experiments presented in C, D, G, and H. Data are shown as mean ± SD and are derived from at least three independent experiments. NC, negative control; PC, positive control. *P < 0.05; **P < 0.01.

Fig. 6.

The SP110 transgene is heritable and is expressed in offspring macrophages. (A and B) Southern blot analysis of three offspring cattle using probe 1 (A) and probe 2 (B). The results show that one of the offspring is heterozygous for the SP110 knockin. Lanes 1–3 represent the three offspring cattle. NC, negative control; PC, positive control. (C) The expression level of SP110 was detected by Western blots (WB). Lanes 1–3 represent the three offspring cattle. GAPDH serves as a loading control. (D) In vitro multiplication of M. bovis in the macrophages from control cattle, control offspring, or heterozygous offspring. M. bovis multiplication was determined by cfu assay. Control offspring are the two offspring cattle without the SP110 transgene. (E) Apoptosis and necrosis rates of control and offspring macrophages infected with M. bovis were determined by flow cytometry.