Development of genome engineering technologies in cattle: from random to specific

Abstract

The production of transgenic farm animals (e.g., cattle) via genome engineering for the gain or loss of gene functions is an important undertaking. In the initial stages of genome engineering, DNA micro-injection into one-cell stage embryos (zygotes) followed by embryo transfer into a recipient was performed because of the ease of the procedure. However, as this approach resulted in severe mosaicism and has a low efficiency, it is not typically employed in the cattle as priority, unlike in mice. To overcome the above issue with micro-injection in cattle, somatic cell nuclear transfer (SCNT) was introduced and successfully used to produce cloned livestock. The application of SCNT for the production of transgenic livestock represents a significant advancement, but its development speed is relatively slow because of abnormal reprogramming and low gene targeting efficiency. Recent genome editing technologies (e.g., ZFN, TALEN, and CRISPR-Cas9) have been rapidly adapted for applications in cattle and great results have been achieved in several fields such as disease models and bioreactors. In the future, genome engineering technologies will accelerate our understanding of genetic traits in bovine and will be readily adapted for bio-medical applications in cattle.

Keywords: CRISPR-Cas9; Cattle; Genome engineering technologies; Transgenesis; Transposon.

Fig. 1

Milestones in the production of transgenic cattle

Fig. 2

Representative pictures of oocytes. Left: oocyte from rats, Middle: oocyte from cow, Right: oocyte from pigs. Scale = 50 µm

Fig. 3

Illustration depicting micro-injection (MI) and somatic cell nuclear transfer (SCNT) for genome modified cattle (GMC). MI takes long time for GMC production without mosaicism while SCNT provides one step procedure for GMC

Fig. 4

Illustration depicting genome integration via the piggyBac (PB) transposon. The PB transposase recognizes the PB-long term repeat (LTR) sequences, cuts it, and inserts itself into a “TTAA” sequence in the host genome. The inset represent Hela cells with the PB- green (G)- and red (R)-fluorescent protein (FP) gene linked by 2A peptide sequences

Fig. 5

Pregnancy of cloned embryos derived from tetracycline dependent gene expression. a Illustration of the tetracycline dependent gene expression system in cattle; the somatic cell nuclear transfer protocol was presented in our previous publication [15]. In brief, piggyBac (PB) DNA containing red fluorescence protein (RFP) under tetracycline-controlled transcription activation promoter (tet-on) was transfected into bovine somatic cells with the PB-transposase and -reverse tetracycline-controlled transactivator (rtTA). An RFP expressing cell was microinjected into enucleated bovine oocytes, fused, and activated chemically. The blastocysts were transferred into a recipient cow. b Representative confirmation pictures of pregnancy using ultrasonography (upper) and collected fetuses (lower); c RFP expression following doxycycline treatments; to know if RFP expression was induced by tetracycline, a small piece of tissue was exposed with Doxycycline [Dox (+)] or without Doxycycline [Dox (−)]; d Identification of the transgene integration site via next generation sequencing analysis. Four transgene integration sites were identified

Fig. 6

Illustration of knock-out/-in cattle. SCNT combined with homologous recombination (HR) and genome editing is a useful approach, though it is limited by abnormal reprograming and low success rates. Simple micro-injection of Cas9 and sgRNA for the target region will be useful for the production of genome edited cattle with high efficiency and genomic stability. NHEJ: Non-homologous end joining; HDR: Homology directed repair