Comparative Analysis of piggyBac, CRISPR/Cas9 and TALEN Mediated BAC Transgenesis in the Zygote for the Generation of Humanized SIRPA Rats

Abstract

BAC transgenic mammalian systems offer an important platform for recapitulating human gene expression and disease modeling. While the larger body mass, and greater genetic and physiologic similarity to humans render rats well suited for reproducing human immune diseases and evaluating therapeutic strategies, difficulties of generating BAC transgenic rats have hindered progress. Thus, an efficient method for BAC transgenesis in rats would be valuable. Immunodeficient mice carrying a human SIRPA transgene have previously been shown to support improved human cell hematopoiesis. Here, we have generated for the first time, human SIRPA BAC transgenic rats, for which the gene is faithfully expressed, functionally active, and germline transmissible. To do this, human SIRPA BAC was modified with elements to work in coordination with genome engineering technologies-piggyBac, CRISPR/Cas9 or TALEN. Our findings show that piggyBac transposition is a more efficient approach than the classical BAC transgenesis, resulting in complete BAC integration with predictable end sequences, thereby permitting precise assessment of the integration site. Neither CRISPR/Cas9 nor TALEN increased BAC transgenesis. Therefore, an efficient generation of human SIRPA transgenic rats using piggyBac opens opportunities for expansion of humanized transgenic rat models in the future to advance biomedical research and therapeutic applications.

Engineering multicellular systems: Using synthetic biology to control tissue self-organization

“…Synthetic DNA of even very large size can now be produced relatively cheaply and landing pads can be used to integrate these larger synthetic constructs into the genome in site-specific ways [11]. Entirely artificial chromosomes can also be used as carriers of exogenous DNA, either alone or in combination with transposon-based technology [12]. In addition, advances in CRISPR based technologies have allowed for unprecedented manipulation of endogenous loci allowing both genetic replacement and nuanced gene control [13].…”

Porcine signal regulatory protein alpha binds to human CD47 to inhibit phagocytosis: Implications for human hematopoietic stem cell transplantation into severe combined immunodeficient pigs

“…Together, these results show that the porcine SIRPA‐human CD47 binding interaction was sufficient to inhibit phagocytosis of human cells. Mouse and rat models have previously required genetic modification to express human SIRPA to allow engraftment of human cells . Due to the fact that porcine SIRPA binds to human CD47 intrinsically, we do not believe that pigs would require introduction of the human SIRPA gene to allow for HSC engraftment.…”

“…The SIRPA protein in C57BL/6 and BALB/c both have low affinity to human CD47 that is not sufficient for inhibitory signaling, which explains the lack of successful human cell engraftment in these mouse strains, and further demonstrates the importance of SIRPA‐CD47 recognition for humanization of these rodent models . Certain rats and mice of mixed 129/BALB/c background are genetically modified to express human SIRPA, making these animals candidates for humanization if crossed onto a SCID genetic background . Interestingly, past studies have investigated the interaction between porcine CD47 and human SIRPA, the opposite interaction to that described here, in relation to transplantation of pig organs into human patients.…”

SENCRstabilizes vascular endothelial cell adherens junctions through interaction with CKAP4

“…Generation of Humanized SENCR Mice. A BAC clone (RP11-744N12) was acquired from the Children's Hospital Oakland Research Institute to produce humanized SENCR mouse lines, as described previously (42). Briefly, a BAC vector (pBACe3.6) was modified into RP11-744N12 (227,863 bp) containing the human SENCR and FLI1 gene loci by recombineering.…”