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.
Highlights
Mammalian model systems provide an essential platform in biomedical research for deciphering the complexities underlying the pathogenesis of human disease, and for developing the applicative and translational potential of new therapies
The selected human SIRPA containing Bacterial artificial chromosomes (BACs) was retrofitted with a cassette containing the 5′and 3′piggyBac Terminal Inverted Repeat (TIR) elements flanking the spectinomycin resistance gene, by replacing the chloramphenicol resistance gene in the vector backbone (Fig. 1A)
In the negative control experiment, 25 embryos (E15) were genotyped (32.1% relative to the number of embryos transferred), of which 1 embryo was found to be PCR positive (1.3% relative to the total number of embryos transferred) (Table 1). These findings suggest that: 1) co-injecting hSIRPA-BAC-terminal inverse repeat (TIR) DNA with hyPBase piggyBac increases the number of pups that are PCR positive for the presence of hSIRPA; and 2) while increasing the amount of hyPBase co-injected with 100 ng may give rise to higher number of pups relative to the number of embryos injected, the genotyping result indicates a higher percentage of PCR positive pups relative to the total number of pups when the amount of hyPBase injected is decreased to 25 ng
Summary
Mammalian model systems provide an essential platform in biomedical research for deciphering the complexities underlying the pathogenesis of human disease, and for developing the applicative and translational potential of new therapies. To confirm that the enhanced engraftment of human cells in NOD strains is dependent on polymorphic SIRPα, thereby ruling out the possibility of causation or influence by strain specific extraneous factors, Strowig et al.[20] generated human SIRPA BAC transgenic mice on mixed 129/BALB/c background, and demonstrated that the expression of human SIRPA enhanced human cell engraftment and improved functionality of human adaptive immune system in vivo For these reasons, an efficient method for generating human SIRPA BAC transgenic rats would allow for the building of a repository of humanized SIRPA rats on various immune-deficient rat strains[21], for use as a tool for studying the engraftment potential of human cells and tissues, as well as for reproducing human immune diseases and evaluating therapeutic strategies. Of relevance to this study is a recent pioneer publication showing that BACs retrofitted with TIR elements can be efficiently transposed in mouse zygotes[34]
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