Generation of mastitis resistance in cows by targeting human lysozyme gene to β-casein locus using zinc-finger nucleases

Abstract

ResearchCite this article: Liu X et al. Mastitis costs the dairy industry billions of dollars annually and is the most consequential disease of dairy cattle. Transgenic cows secreting an antimicrobial peptide demonstrated resistance to mastitis. The combination of somatic cell gene targeting and nuclear transfer provides a powerful method to produce transgenic animals. Recent studies found that a precisely placed doublestrand break induced by engineered zinc-finger nucleases (ZFNs) stimulated the integration of exogenous DNA stretches into a pre-determined genomic location, resulting in high-efficiency site-specific gene addition. Here, we used ZFNs to target human lysozyme (hLYZ) gene to bovine b-casein locus, resulting in hLYZ knock-in of approximately 1% of ZFN-treated bovine fetal fibroblasts (BFFs). Gene-targeted fibroblast cell clones were screened by junction PCR amplification and Southern blot analysis. Gene-targeted BFFs were used in somatic cell nuclear transfer. In vitro assays demonstrated that the milk secreted by transgenic cows had the ability to kill Staphylococcus aureus. We report the production of cloned cows carrying human lysozyme gene knock-in b-casein locus using ZFNs. Our findings open a unique avenue for the creation of transgenic cows from genetic engineering by providing a viable tool for enhancing resistance to disease and improving the health and welfare of livestock.

Transgenesis in farm animals-A review

“…It recognizes and binds DNA at specific site with the help of DNA binding domain and produce cut thus causing double stranded break (DSB) in DNA at a specific location due to endonuclease domain present followed by transferring of external DNA by induction of homology directed repair or non-homology terminal joining (Miao, 2012). ZFN-mediated genome editing has shown good results in mice, rats, human and recently, in 2014, mastitis resistant transgenic calves have been produced using ZFN (Liu et al, 2014). Even though ZFNs have been used for targeted genome editing in various organisms, the major limitations faced with ZFN are limited modularity due to context dependent DNA-binding effects, making difficult for ZFNs to target any desired DNA sequence (Ramirez et al, 2018).…”

Invited review: Reproductive and genomic technologies to optimize breeding strategies for genetic progress in dairy cattle

“…Many considered uses of the technology are the same as those already considered in current breeding programs including production, health, and welfare, but with targeted efforts on known causative variants. Examples of edited dairy cattle include cattle with increased resistance to tuberculosis (Wu et al, 2015), the knockout of the betalactoglobulin gene , and enhanced mastitis resistance (Liu et al, 2013(Liu et al, , 2014. A promising first use of gene editing in the dairy industry to address welfare issues may be the production of hornless dairy cattle with the introduction of the POLLED allele, which is nearly fixed in some beef breeds but low in frequency in Holsteins (Carlson et al, 2016).…”

Genome Editing with Engineered Nucleases in Economically Important Animals and Plants: State of the Art in the Research Pipeline

“…Mastitis is a bacterial infection of mammary gland in the udder and is among the most common and costly diseases in dairy cattle. Liu et al (2014) used ZFNs to knock-in the human lysozyme gene (hLyz) so that the transgenic cows would produce the lysozyme in the milk and thus become resistant to the bacteria causing the disease. Five transgenic calves were produced whose mammary glands, after infusion by viable bacterial cultures, remained uninfected and no systemic response was observed even after blood analysis.…”

“…In the already mentioned study by , the human lactoferrin gene was knocked in using TALENs. Liu et al (2014) and Lee et al (2014) have knocked in the human erythropoietin (hEpo) gene in the β-casein gene locus in cattle using TALENs. The β-casein gene locus was also targeted in the study by Liu et al (2014) who focused on mastitis in cows.…”

“…Liu et al (2014) and Lee et al (2014) have knocked in the human erythropoietin (hEpo) gene in the β-casein gene locus in cattle using TALENs. The β-casein gene locus was also targeted in the study by Liu et al (2014) who focused on mastitis in cows. Mastitis is a bacterial infection of mammary gland in the udder and is among the most common and costly diseases in dairy cattle.…”