The production of therapeutic recombinant proteins in heterologous systems has gained significance since the last decade. For recombinant proteins that require post-translational modifications (PTMs), mammalian systems are preferred. Chinese hamster ovary (CHO) cells are the mammalian cells of choice for production of recombinant proteins. This is because of their ability to provide correct protein-folding and post-translational modifications, displaying high productivity at large scale, ability to grow in suspension mode at high densities in a serum-free media, incapable of infection by most viruses and their history of regulatory approvals. There is an established state of the art technology for development of CHO cells for recombinant protein production. This technology relies on random integration of the gene of interest and gene amplification process for obtaining high expressing clones. There is a high degree of clonal heterogeneity and instability observed in the screened clones. To overcome the process of random integration, this report describes a lentivirus based screening for search of stable and high expressing integration sites in CHO cells. The integration sites are identified by using nrLAM-PCR (non-restrictive linear amplification mediated PCR) coupled with high throughput sequencing. Lentivirus are chosen as they preferentially integrate within the coding regions rendering the possibility of obtaining stable and high expressing clones. In addition, lentivirus vector is designed to possess landing pad for recombinase mediated cassette exchange of viral sequence with foreign DNA. The report describes a successful cassette exchange reaction but with low efficiency. Genome engineering technologies such as CRISPR/Cas, TALENs can used for targeted gene insertion at integration sites and thus establishing stable and efficient production of recombinant proteins in CHO cells. Additionally, an approach for designing synthetic promoters based on Ef1α promoter architecture has been shown. Synthetic promoters are useful for expression of multi-gene cassettes as they are short in length and provide comparable expression levels to the native mammalian promoter.

There is a vast diversity in tetrapod lung branching morphology. Phylogenetically, much of the pulmonary diversity among vertebrates appears to arise from the way epithelial tubes branch or form saccular (cyst) structures. Fgf10 activity has been shown to play a critical role in regulating branch versus cyst morphology. We hypothesize that the species-specific differences in lung morphology may be primarily due to species-specific differences in Fgf10 expression. To test this hypothesis, we have performed bioinformatic analyses on the Fgf10 locus and have identified a conserved 11 kb noncoding region that potentially contains the Fgf10 lung enhancer. We are taking a large DNA sequence upstream of the Fgf10 gene of the American Alligator and swapping it into the orthologous locus in the genome of chicken primordial germ cells (cPGCs). We are accomplishing these swaps by using a combination of homology directed repair (HDR) and recombinase mediated cassette exchange (RMCE) in cPGCs. These edited cell lines can be used to generate germline chimeric chickens capable of producing offspring that putatively drive Fgf10 expression in the lung under control of regulatory sequences from various other reptiles. We have also generated a cPGC line where, through RMCE, we can easily target any enhancer from any organism to drive a GFP reporter as a means to test the temporal and spatial regulatory characteristics of these enhancers. This work is funded through a BYU Turkey Vaccine Grant and a Skaggs Mentoring Grant.

The chicken is a classic model organism that has provided key insights into embryonic development. Chicken embryos can be directly manipulated and observed during development while retaining the potential to reach adulthood. Despite this benefit, the utility of the chicken in studying development has been limited by the difficulty of introducing genetic changes to the genome. The recent development of cell culture conditions for chicken primordial germ cells (cPGC) has made it feasible to produce transgenic chickens, but there is still a lack of tools for introducing genetic modifications into cPGCs. Recombinase Mediated Cassette Exchange (RMCE) is a technique that has been utilized in traditional genetic systems to generate multiple alleles at a given locus but has not yet been adapted to the chicken. In order to use RMCE in the chicken, we inserted Lox sites into cPGC using CRISPR/Cas9. We targeted the ovalbumin locus and potential genomic safe harbor sites (GSH) identified using genomic data. We performed RMCE to exchange green fluorescent protein (GFP) into these loci. We observed RMCE efficiency as less than 1% at each loci. We then designed a system using a drug inducible Caspase 9 (iCasp9) to select for cells that underwent cassette exchange. This method enabled us to obtain a population of 100% edited cells. We anticipate that this tool will increase the utility of the chicken as a model organism, livestock, and bioreactor.