Genome editing via CAS9-mediated DNA strand breaks permits the rapid and accurate germline genetic modification of murine tumor models. Previous results from our laboratory demonstrate that G-CSF mediated activation of pSTAT3 is critical for the survival and function of a small tumor subpopulation expressing the G-CSF receptor (CD114). These cells are highly tumorigenic, self-renew and generate differentiated progeny in vivo, fulfilling the accepted definition for cancer stem cells. Furthermore, G-CSF regulates important interactions among immune subsets, stroma and tumor within the microenvironment. G-CSF Knockout mice (CSF3-/-) are viable, reproductive, and have intact immune systems with mild neutropenia.
To further evaluate the functional significance of G-CSF signaling in murine MYCN-driven neuroblastoma, which is highly strain dependent, we sought to directly knockout CSF3-/- in the pTH-MYCN (SVJ/129) transgenic model of NB via CRISPR/CAS9 targeted deletion. As the CSF3 genomic region spans only 3,317 bps, we deigned two guide sequences targeting 5’ of exon 1 and 3’ of exon 5 within the 3’UTR to excise the entire coding sequence (exons 1-5) for embryonic stem cell injection. Blastocysts harvested from super-ovulated female pTH-MYCN+/- mice mated to wild-type stud males were injected with both guide RNAs as well as CAS9 mRNA, and transferred into pseudo-pregnant surrogate females. Genomic PCR based genotyping demonstrated three founder mice with the following phenotypes: (CSF3-/+, MYCN+/+ Female), (CSF3-/+, MYCN+/- Male), (CSF3-/+, MYCN+/- Male). DNA sequencing confirmed knockout of the coding sequence on one allele in each founder. These mice are currently breeding to generate large cohorts with the following phenotypes: (CSF3-/-, MYCN+/+), (CSF3-/+, MYCN+/+), CSF3+/+, MYCN+/+).
These genome edited immunocompetent transgenic mice will permit us to directly evaluate the impact of G-CSF deficiency on tumor incidence, prevalence, and rate of progression in murine model of aggressive neuroblastoma. Administration of exogenous G-CSF will permit in vivo phenotypic rescue in G-CSF-/- mice.