Browsing by Subject "Allele introgression"

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    Genome engineering in large animals for agricultural and biomedical applications
    (2013-08) Tan, Wenfang
    Precision genetics will enhance genome-based improvement of livestock for agriculture and biomedicine. This thesis aimed to modify large animal genomes with precision; as the technologies progressed, our capability expanded from random insertional transgenesis to nucleotide-level precision. It began with Sleeping Beauty (SB) transposon mediated rapid integration of dominant negative Myostatin alleles. All piglets generated from treated cells harbored the transgenes; however, we were unable to study phenotypes due to death of the founder animals. We then sought to introgress a SNP into porcine Myostatin through recombinant Adeno-associated Virus (rAAV) mediated gene targeting. We achieved a 2x10-4 targeting frequency but only one-half of the targeted colonies harbored the SNP. Similarly, we succeeded in porcine LDLR gene knockout; however, targeted clones were often confounded by "bystander" cells with only random insertions of the targeting vector. We turned to develop TALENs for efficient targeting of important genes. TALENs demonstrated high activity in both cultured primary fibroblasts and early stage embryos. A simple SB transposon based co-selection strategy enabled enrichment for TALEN modified cells and efficient isolation of modified clones: single gene mono- and bi-allelic modification was induced in up to 54% and 17% of colonies respectively. It also enabled isolation of colonies harboring large chromosomal deletions (10% of colonies) and inversions (4%) after treatment with two TALEN pairs. We derived miniature swine models of familial hypercholesterolemia from LDLR mono- and bi-allelic TALEN-knockout fibroblasts. We next utilized TALEN and CRISPR/Cas9 stimulated homology-directed repair (HDR) to edit genes with oligonucleotide, plasmid, and rAAV templates without any drug selection. We first introgressed a bovine POLLED allele into horned dairy bull fibroblasts to circumvent manual dehorning. We also introduced single-nucleotide alterations or small indels into 14 additional genes in pig, cattle and sheep, into 10-50% of cells from fibroblast populations treated with TALEN mRNA and oligonucleotides. Up to 67% of propagated colonies harbored the intended edits and over one-half were homozygous. Some edits were naturally occurring SNP alleles, equivalent to non-meiotic inter- or intra-species introgression of valuable alleles. We created pig models for infertility and colon cancer from colonies with TALEN-HDR knockout alleles in DAZL and APC.