Identification <i>AAVS1</i>-like locus from the porcine genome and site-specific integration of recombinase-mediated cassette exchange using CRISPR/Cas9

Gene integration at site-specific loci is a critical approach for understanding the function of a gene in cells or animals. The AAVS1 locus is a well-known safe harbor for human and mouse studies. In this study, we found an AAVS1-like sequence (pAAVS1) in the porcine genome using the Genome Browser and designed TALEN and CRISPR/Cas9 to target the pAAVS1. The efficiency of CRISPR/Cas9 in porcine cells was superior to that of TALEN. We added a loxP-lox2272 sequences to the pAAVS1 targeting donor vector containing GFP for further exchange of various transgenes via recombinase-mediated cassette exchange (RMCE). The donor vector and CRISPR/Cas9 components were transfected into porcine fibroblasts. Targeted cells of CRISPR/Cas9-mediated homologous recombination were identified by antibiotic selection. Gene knock-in was confirmed by PCR. To induce RMCE, another donor vector containing the loxP-lox2272 and inducible Cre recombinase was cloned. The Cre-donor vector was transfected into the pAAVS1 targeted cell line, and RMCE was induced by adding doxycycline to the culture medium. RMCE in porcine fibroblasts was confirmed using PCR. In conclusion, gene targeting at the pAAVS1 and RMCE in porcine fibroblasts was successful. This technology will be useful for future porcine transgenesis studies and the generation of stable transgenic pigs.</p

Efficient <i>PRNP</i> deletion in bovine genome using gene-editing technologies in bovine cells

<p>Even though prion (encoded by the <i>PRNP</i> gene) diseases like bovine spongiform encephalopathy (BSE) are fatal neurodegenerative diseases in cattle, their study via gene deletion has been limited due to the absence of cell lines or mutant models. In this study, we aim to develop an immortalized fibroblast cell line in which genome-engineering technology can be readily applied to create gene-modified clones for studies. To this end, this study is designed to 1) investigate the induction of primary fibroblasts to immortalization by introducing <i>Bmi-1</i> and <i>hTert</i> genes; 2) investigate the disruption of the <i>PRNP</i> in those cells; and 3) evaluate the gene expression and embryonic development using knockout (KO) cell lines. Primary cells from a male neonate were immortalized with <i>Bmi-1</i>and <i>hTert</i>. Immortalized cells were cultured for more than 180 days without any changes in their doubling time and morphology. Furthermore, to knockout the <i>PRNP</i> gene, plasmids that encode transcription activator-like effector nuclease (TALEN) pairs were transfected into the cells, and transfected single cells were propagated. Mutated clonal cell lines were confirmed by T7 endonuclease I assay and sequencing. Four knockout cell lines were used for somatic cell nuclear transfer (SCNT), and the resulting embryos were developed to the blastocyst stage. The genes (<i>CSNK2A1, FAM64A, MPG and PRND</i>) were affected after <i>PRNP</i> disruption in immortalized cells. In conclusion, we established immortalized cattle fibroblasts using <i>Bmi-1</i> and <i>hTert</i> genes, and used TALENs to knockout the <i>PRNP</i> gene in these immortalized cells. The efficient <i>PRNP</i> KO is expected to be a useful technology to develop our understanding of <i>in vitro</i> prion protein functions in cattle.</p

Additional file 1: of Long-term health and germline transmission in transgenic cattle following transposon-mediated gene transfer

Raw data of blood analysis from transgenic cattle. (XLSX 15 kb

Additional file 3: of Long-term health and germline transmission in transgenic cattle following transposon-mediated gene transfer

Figure S2. Detection of the expression of YFP in milk from SNU-SB-1 by confocal microscopy. Images of milk from wild type cattle (left) and SNU-SB-1 (right) taken using a high-throughput confocal microscope. YFP: YFP field. (PNG 1685 kb

Additional file 4: of Long-term health and germline transmission in transgenic cattle following transposon-mediated gene transfer

Figure S3. 5′ junction sequence analysis of all integration sites in SNU-F1–1. Sequences showing the genome-to-transposon junctions in the genome of SNU-F1–1 and the integration of transgenes at TTAA sites. (PNG 465 kb

Additional file 7: of Long-term health and germline transmission in transgenic cattle following transposon-mediated gene transfer

Figure S5. Overview of genomic variation in SNU-F1–2. Reference chromosomes from bt1 to btX are denoted by colored boxes at the outer edge. Plots denoting copy number variation (CNV; black dot plots in the green area), coverage (green line plot in the green area) and SNP density (orange histogram in orange area) for the SNU-F1–2 genome are shown for each 10 kb window. (PNG 1244 kb