Production Of Transgenic Cow Using Somatic Gene Targeting With ZFNs

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 double-strand break induced by engineered zinc-finger nucleases (ZFNs) stimulated the integration of exogenous DNA stretches into a predetermined genomic location, resulting in high-efficiency site-specific gene addition. Zinc-finger nickases (ZFNickases) are a type of programmable nuclease that can be engineered from zinc-finger nucleases to induce site-specific single-strand breaks or nicks in genomic DNA, which result in homology-directed repair. Whereas zinc-finger nuclease-mediated gene disruption has been demonstrated in pigs and cattle, they have not been used to target gene addition into an endogenous gene locus in any large domestic species.The aim of this study was to perform efficient and reproducible gene targeting in bovine fetal fibroblasts by inserting the exogenous human lysozyme gene (hLYZ) and lysostaphin gene (Lys) into the beta-casein locus using engineered ZFNs and ZFNickases so that the exogenous antimicrobial protein could be produced in the mammary gland of the gene-targeted cow. The main contents of this research are as follows:1.Assessment of ZFN activity in bovine fetal fibroblastsDifferent amounts of plasmids expressing of ZFNs were co-transfected with pEGFP-C1into BFFs. The frequency of ZFN-mediated disruption at the target site in each pool of cells was determined using the CEL-I nuclease assay. To determine the molecular identity of the ZFN-mediated mutations in the beta-casein gene (CSN2) easily and quickly, we amplified the CSN2ZFNs target region from genomic DNA that was harvested3days after BFFs were transfected with ZFNs. PCR products corresponding to the targeted site were sequenced directly.2.Efficient ZFN-induced hLYZ integration into the CSN2locus in BFFs To investigate the efficient ZFN-induced exogenous gene integration into a native chromosomal locus, we introduced the targeting vector pCSN2-hLYZ-Neo-GFP into BFFs along with expression plasmids encoding designed ZFNs that were engineered to introduce a DSB in intron2of CSN2.Stably transducedcells that were present in target cell populations were identifiedfollowing7-9days of (600μg/ml) G418selection.Stably transfected cell clones were digested using trypsin and transferred to a48-well plate and cultured for PCR detection.We found that15.8%(84) of the G418-resistant colonies contained correctly targeted cells.3.The expression of human lysozyme cDNA and EGFP reporter gene in BMECs in vitroHuman lysozyme-containing constructs pEGFP-C-hLYZ, pEGFP-S-hLYZ and pEGFP-I-hLYZ were sequentially assembled and evaluated following their transfection into third-passage BMECs. The transfected cells with pEGFP-C-hLYZ and pEGFP-S-hLYZ were all observed to have EGFP fluorescence48h post-transfection. BMECs transfected with pEGFP-I-hLYZ along with pZFN1/2were observed to have EGFP fluorescence48h post-induction.4.Mutation of the ZFN catalytic domain generates a ZFNickaseTo generate ZFNs with strand-specific nicking activity, we mutated the Fokl catalytic domain in one of the two ZFNs necessary for dimerization and subsequent DNA cleavage. These data demonstrate that elimination of cleavage activity in one half of a ZFN pair by the introduction of a D450A mutation results in the generation of a potent, strand-specific ZFNickase.5. ZFNickases promote HDR-mediated site-specific gene additionWe constructed the gene-targeting vector, pCSN2-Lys-Neo-EGFP, by inserting the mature portion of the lysostaphin gene with splice acceptor sequence into the vector pTCSN2. We introduced this targeting vector along with expression plasmids encoding ZFNs/ZFNickases engineered to introduce a DSB/SSB in intron2of CSN2into BFFs. We found that8.7%(99) of the G418-resistant colonies contained correctly targeted cells.6. Somatic cell nuclear transfer and cloned transgenic cow productionGene-targeted cloned embryos were produced from bovine oocytes of ovaries collected from slaughtered cows.Three cell lines targeted human lysozyme gene from the six targeted colonies suitable for NT were selected as donor cells. The developed cloned embryos and were transferred to118recipient cows, and finally,5transgenic calves were born.12cell lines targeted lysostaphin gene (6colonies from ZFNickases-induced group and6colonies from ZFNs-induced) without random integration of them were prepared for somatic cell nuclear transfer. Ultimately,19of140pregnancies developed to term (16derived from the ZFNickase-induced group and3from the ZFN-induced group), yielding14live gene-targeted calves, with birth weights between45and55kg. Six of these calves perished soon after birth but eight lived for more than1month.7. Analysis of cloned cowsTissue was recovered from the cloned cows for both junction PCR and Southern blot analysis. The two junction PCR screens for each locus revealed patterns consistent with targeting in all the targeted samples that were recovered. In Southern blot analyses, both5’(external) and hLYZcoding sequence or lysostaphin sequence (internal) probes hybridized to restriction fragments of the correct size.These data show that cows carrying targeted gene additions can be generated by nuclear transfer.To produce milk containing human lysozyme or lysostaphin from transgenic cows in the context of the original composition without any changes, we did not deliberately select homozygous integrants.Milk from transgenic cows was tested against a variety of microorganisms. The transgenic cows’ability to resist infection by Sta. aureus, E. coli or Str. agalactiaewas tested by intramammary infusion of viable bacterial cultures.All of above results indicated that ZFNs/ZFNickase-treated cells can be successfully used in somatic cell nuclear transfer, resulting in live-born gene-targeted cows. Furthermore, the gene-targeted cows secrete human lysozyme and lysostaphin in their milk and in vitro assays demonstrate the milk’s ability to kill Staphylococcus aureus. Our success with this strategy will facilitate new transgenic technologies beneficial to both agriculture and biomedicine.