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Definitions

• GEMMs Genetically engineered mouse models
• EP electroporation-mediated
• viral gene deliveries are increasingly employed as a more rapid means of creating somatic mosaics, such as modeling tumors. Both techniques can virtually target anywhere in the body.
• TRE tetracycline-regulated system
• shRNAs PB-EP
• PB-EP and viral methods have many pitfalls. Viral vectors have limited payloads, incite immune responses, and require complex preparation expertise, and both PB-EP and viral delivery suffer from their unpredictable genomic integration patterns, subsequent insertional mutagenesis, and epigenetic transgene silencing. Most importantly for interrogation of gain-of-function (GOF) mutations, both non- GEMM techniques result in clonal genotypic/phenotypic variability, often caused by transgene copy number variation (CNV) or chromosomal-positional variability, whereas GEMMs ensure constant gene dosage and zygosity quantified during mouse engineering. Therefore, non-GEMM-based evaluation of GOF protein functions is often confounded by such supraphysiological phenomena as overexpression artifacts, unintended cytotoxicity, and transcriptional squelching.
• CNV transgene copy number variation
• GEMMs ensure constant gene dosage and zygosity quantified during mouse engineering. Therefore, non-GEMM-based evaluation of GOF protein functions is often
• FIG. 1 (a-f) shows that dRMCE in heterozygous mTmG generates three recombinant lineages in vivo and in vitro in accordance with various embodiments of the present invention
• a) Flp-Cre expression vector catalyzes either Cre-mediated excision or dRMCE on Rosa26m TmG allele in the presence a dRMCE donor vector, resulting in two distinct recombinant products
• b) Nucleofection of heterozygous Rosa26 WT/mTmG mNSCs result in three possible lineages: tdTomato+, EGFP+, and TagBFP2+.
• Figure 2 (a-g) shows that rapid generation of somatic mosaics using in vivo dRMCE can be used for autochthonous tumor modeling in accordance with various embodiments of the present invention
• Figure 3 depicts measurement of dRMCE efficiency in heterozygous mTmG mNSCs by FACS analysis, and confirmation of correct protein translation at non- clonal population level in accordance with various embodiments of the present invention
• b) FACS analysis indicates the approximate dRMCE efficiency in neural stem cells, and no obvious difference between Flp- 2A-Cre and Flp-IRES-Cre in their catalytic efficiencies
• Western blot indicating normal transgene production from non-clonal aggregate cells and lack thereof in FACS negative population. Removal of tdTomato expression is also observed.
• Figure 4 (a-g) depicts dRMCE-compatible inducible donor construct that can be used to interrogate overexpression and GOF mutations of genes in accordance with various embodiments of the present invention
• a) dRMCE-compatible TRE plasmid b) Heterozygous mTmG mNSCs are nucleofected with plasmid in a), treated with puromycin, and turned into a colorless population.
• Scale bars ⁇ c) Induction of EGFP expression in the cell line that constitutively express rtTA-VlO-AUl .
• Figure 5 depicts PCR screening and western blot analysis confirming dRMCE-mediated excision of tdTomato cassette and integration of donor cassette at Rosa26mTmG locus in accordance with various embodiments of the present invention
• b) PCR screening analysis reveals that rtTA-VlO-AUl cassette is correctly integrated downstream of C AG-promoter in cells that are resistant to puromycin treatment
• c Western blot analysis of the cell line from Fig.
• Figure 6 (a-c) shows that in vivo dRMCE in VZ/SVZ is detectable at 2 days and stable at 2 weeks post-EP with subsequent removal of membrane-tdTomato expression in accordance with various embodiments of the present invention
• Scale bars ⁇ ; Inset: 20 ⁇ .
• Figure 7 (a-c) shows that Hras G12V confers gene-dosage-dependent differential phenotypes in accordance with various embodiments of the present invention
• Figure 8 (a-b) depicts examination of Hras Gi -recombined mTmG cells in the striatum in accordance with various embodiments of the present invention
• a) Two weeks post-EP shows clear lineage divergence between EGFP+ cells that underwent Cre-mediated excision of tdTomato cassette and HrasG12V+ cells with successful dRMCE.
• Figure 9 (a-b) depicts control electroporation experiments for in vivo dRMCE in accordance with various embodiments of the present invention
• Figure 10 (a-e) depicts knockdown of Nfl, Pten, and Trp53 mRNAs by qPCR, and the dormancy of these mutations in neuronal lineage in accordance with various embodiments of the present invention
• Olfactory bulb neurons stably expressing TagBFP2-multi-miR-E at 6 months post-EP shows no sign of aberrant transformation.
• Scale bar 200 ⁇
• Figure 11 (a-j) depicts confirmation of Pdgfra and V5-tagged Trp53 expression by in vitro and in vivo immunohistochemistry in accordance with various embodiments of the present invention
• a) In vitro assessment of transgene expression after dRMCE in heterozygous mTmG mNSCs shows the coexpression of nuclear EGFP with Pdgfra, V5 (Trp53R270H), and P53. Note the presence of contaminating mG cells with membrane EGFP and no tdTomato or transgene expression. Scale bars: 50 ⁇ .
• Atrx is expressed in the majority of EGFP+ cells in K27M tumors. A small subset of EGFP+ cells (yellow arrows) has lost Atrx antigenicity, e) G34R cells at 100 days post-EP express Atrx. f) CRISPR/Cas9 targeting leads to highly efficient loss of Atrx in EPed cells, g) Cortically-infiltrating G34R tumor at 120 days post-EP.
• K27M tumor at 120 days post-EP is predominantly sub-cortical
• i-1 shows staining pattern disparity at tumor margin
• j) A monoclonal antibody demonstrates that expression of H3f3a transgene is consistent throughout Rosa26H3f3a-K27M/Pdgfra/Trp53 tumor. *-channel pseudocolored green from Cy5 wavelength for increased contrast.
• Figure 12 depicts alternative reporter mice amenable to in vivo MADR or
• MADR MAX and extension of the method to Ribotrap and IKMC repository mouse lines with gene-trap alleles in accordance with various embodiments of the present invention.
• CAG-based reporter mice that are similar to mTmG mice in construction and compatible with in vivo MADR to achieve mutant lineage tracing studies or orthogonal RNA isolation using Ribotrap heterozygotes. Additionally, this method can extend to thousands of gene-trap mice that, as an example, flank loxP and FRT around important exons. in vivo MADR at such loci would enable 1) lineage tracing of heterozygous/homozygous null cells at the locus, as well as 2) swapping the locus with a transgene.
• loxP core GCATACAT (SEQ ID NO: 12); minimal FRT: GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC (SEQ ID: 13); second panel: minimal FRT: GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC (SEQ ID: 13); loxP core: ATAGTATGC (SEQ ID: 14).
• Figure 13 (a-c) depicts examination of HrasG12V-recombined mTmG cells in the striatum in accordance with various embodiments of the present invention.
• A Two weeks post-EP shows clear lineage divergence between EGFP+ cells that underwent Cre- mediated excision of tdTomato cassette and HrasG12V+ cells with successful MADR. Scale bars: ⁇ .
• B As low as 10 ng/ ⁇ recombinase-expression vector in EP mixture can catalyze MADR in vivo. Scale bars: ⁇ .
• Figure 14 (a-g) shows that generation of MADR glioma models utilizing recurrent mutations observed in pediatric GBM yields phenotypes consistent with human subtypes and gives insights into alterations H3f3a PTMs in accordance with various embodiments of the present invention.
• Inset B-l shows a lack of significant cortical infiltration.
• Rosa26H3f3aG34R/Pdgfra/Trp53 exhibits a glial hyperplasia in the striatum and a small mass of EGFP+ cells in the ventral forbrain medial to the piriform cortex.
• D) Rosa26H3f3aG34R/Pdgfra/Trp53 EGFP+ tumor cells are hypomethylated at H3K27.
• E) EGFP+ tumor cells exhibit variable H3f3a serine 31 phosphorylation at tumor margin versus the core.
• F-G) Zoom-in images of regions E) show that EGFP+ nuclear phosphorylated- serine 31 expression is higher in the tumor margin and attenuated in the core despite the increase in overall cell density in the core.
• Figure 15 (a-h) shows that rapid generation of somatic mosaics using in vivo
• MADR-incorporated Cas9 and PCR-derived sgRNAs allows for interrogation of transdifferentiation within glioma in accordance with various embodiments of the present invention.
• Nfll Exon 42 AACTCCCTCGATGTGGCGGCTCATCTGCCC (SEQ ID NO: 15); AACTCCCTCGAATGTGGCGGCTCATCTGCCC (SEQ ID NO: 16); Trp53 Exon 2 TCTCCTGGCTCAGAGGGAGCTCGAGGCTG (SEQ ID NO: 17); TCTCnnnnnnAGAGGGAGCTCGAGGCTG (SEQ ID NO 18).
• Glioma cells are largely 01ig2+ with small pockets of heterogeneity (white arrow).
• G) Pdgfrb immunostaining of brain demonstrates that most pericytes are non-tumor derived (i.e. tdTomato+; Ei).
• G 3- 5 Single z plane from G 2 and subsequent englargment thereof demonstrates the lack of colocalization of tdTomato and Pdgfrb (arrowhead).
• H Des (aka Desmin) immunostaining of brain pericytes.
• Hi Clusters of Des+ pericytes which do not colocalize with tdTomato.
• H 2 Single z plane from projection seen in F 1 . *-channel pseudocolored green from Cy5 wavelength for increased contrast
• FIG. 16 (a-f) depicts investigation of transdifferentiation in MADR glioma in accordance with various embodiments of the present invention.
• C-D 2 Rare tdTomato- /Pecaml+ figures can be observed but do not link up to vasculature.
• control elements refers collectively to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control elements need always be present, so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.
• promoter region is used herein in its ordinary sense to refer to a nucleotide region including a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence.
• operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
• control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
• the control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
• intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
• Rosa26mTmG is a widely used reporter line that constitutively expresses membrane tdTomato and switches to EGFP expression upon Cre-mediated excision of tdTomato cassette.
• dRMCE dRMCE in mTmG
• we utilized the unused blue fluorescence channel and created a promoter-less donor plasmid encoding TagBFP2, as well as TagBFP2-tagged HrasG12V, flanked by loxP and FRT sites Fig. Id and Fig. 2a.
• Both mTmG and TagBFP2 plasmid contain minimal 34-bp FRT, which is refractory to Flp- mediated integration.
• the open reading frame is preceded by PGK polyadenylation signal (pA) and trimerized SV40 pA that will preempt spurious transcription from unintegrated episomes and randomly integrated whole-plasmids, which is known to occur with electrochemical transfections.
• the ORF is followed by woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), which increases transgene expression and a rabbit beta-globin pA, which efficiently terminates transcription (Fig. la).
• WPRE woodchuck hepatitis virus post-transcriptional regulatory element
• mNSC mouse neural stem cell line
• dRMCE with TagBFP2 donor plasmid and Flp-Cre expression vector gave rise to three possible results, with cells remaining tdTomato+ or turning green or blue (Fig. lb,c and Fig. 3a).
• FACS analysis indicated the rapid proliferation of HrasG12V cells and also approximate efficiency of dRMCE in mNSCs at around 1% (Fig. 3b).
• 5% of cells positive for blue fluorescence retained either green or red fluorescence, which can be explained by the relatively slow degradation kinetics of membrane tdTomato (Fig. 3b,c).
• PCR screening revealed the perdurance of the EGFP cistron in some cells, but the expression of EGFP is mitigated by the upstream polyA elements and the distance from the CAG-promoter (Fig. 5b). Supporting this, we did not observe EGFP autofluorescence, and western blot of these cells showed EGFP expression only with doxycycline treatment (Fig. 4b and Fig. 5c).
• This in vitro system will be beneficial to interrogating GOF protein functions in various primary cell lines derived from any animal carrying loxP and Frt by providing more homogeneous, inducible stable cell lines.
• As proof-of-principle for this, and to empirically test the utility of the potential leakiness of the 3' cistron of the TRE-Bi element— which could potentially be activated by upstream promoters or enhancers— we also generated a cell line that inducibly expresses the Notch signaling ligand, Dill, with a bicistronic TRE- Bi-Dlll/EGFP donor vector (Fig. 4d).
• All DNA mixtures contained a non-MADR, constitutive nuclear TagBfp2 reporter. Surprisingly, we noted that lowering the donor plasmid concentration to 10 ng/ ⁇ approached nearly 100% MADR MAX efficiency and almost zero Cre-recombined cells with EGFP expression (Fig. If).
• One possible explanation is that increasing the concentration of donor plasmid, hence also of loxP and FRT recombination sites, competes for the recombinases. Alternately, an "overproduction inhibition" mechanism as is seen with transposases is another possibility. All subsequent electroporation mixtures contained 0.5-1 ⁇ / ⁇ 1 of plasmids, in order to generate roughly equivalent numbers of EGFP cells and MADR or MADR MAX cells for side-by-side comparison.
• HrasG12 V mixed with as low as 10 ng/ ⁇ of recombinase expression vector still resulted in aggressive, early- onset tumorigenesis (Fig. 8b).
• Fig. 9 In order to rule out tumor formation due to the expression from randomly integrated or non-recombined episomes, we performed a series of control electroporations (Fig. 9).
• MADM has shown that the combined Trp53- and N/7-LOF mutations promote the pre-malignancy hyperproliferation of oligodendrocyte progenitors (OPCs).
• OPCs oligodendrocyte progenitors
• the system can be modified by creating a donor plasmid carrying spCas9 and delivering it with sgRNAs targeting tumor suppressors.
• EGFP+ sibling cells could serve as a useful control cell population in a manner akin to the wild-type cells in MADM GEMM systems.
• MADM and MASTR allow rigorous, Drosophila-hke deciphering of mutant cells but require de novo GEMM generation depending on the chromosomal location of GOIs.
• Fig. 10B Fig. 10B
• Hras, and Errb2 suggesting GOF and LOF mutations resulting in increased Ras/MAPK signaling may lead to subtly different cell fate alterations.
• Viral and EP tumor models are increasingly employed to study developmental, evolutionary aspects of cancer in various tissue, but there are issues with various gene delivery methods.
• our method could be best suited for in vivo, autochthonous modeling of putative cancer driver genes, and for proof-of-principle, chose Hras G12V , a highly-used activating oncogene. Histological analysis of growth dynamics in putatively single-copy heterozygous mice indicated that Hras G12V cells rapidly overproliferated when compared with EGFP+ populations (Fig. 13A). Moreover, we reasoned that we might be able to distinguish between heterozygous and homozygous populations of cells by breeding the mTmG mice to homozygosity.
• a current unmet need in terms of mouse models of cancer is a higher throughput method of "personalized" tumor modeling.
• platforms that can rapidly and comprehensive model these mutations in vivo in a combinatorial manner.
• H3F3A, PDGFRA, and TRP53 were found to be recurrent in pediatric gliomas.
• the H3F3A mutations were in two residues— K27 and G34.
• resulting patient tumors bearing either K27M or G34R/V mutations in H3F3A exhibit markedly different transcriptomes as well as clinical behaviors.
• H3F3A gliomas of both classes typically harbor TRP53 mutations and can exhibit PDGFRA activating mutations.
• Fig. 14 To model these pediatric tumors in mice, we generated a cassette for co-expression of H3f3a mutations (tagged to EGFP), an activated Pdgfra (D842V), and mutant Trp53 (R270H) (Fig. 14).
• H3f3a, Pdgfra, and Trp53 by immunohistochemistry in vivo and in vitro and noted coincident expression of all proteins (Fig.
• K27M tumors predominantly localized to the sub-cortical structures but cells could be observed in the white matter tracts with a minor amount of cells in the deeper cortical layers (Fig. 11H).
• Fig. 15B Successful targeting in EPed cells was confirmed in tumor population gDNA by sequencing (Fig. 15B). At roughly 5 months we observed terminal morbidity in EPed animals. Pathological analysis led to the diagnosis of glioblastoma multiforme, principally due to the presence of necrosis in the tumor. Immunohistochemically, we observed that the tumor was largely devoid of TdTomato- labeled populations with the exception of vasculature (Fig. 15C-Ci). A small EGFP population was observed near where the original targeting site was expected to reside (Fig. 15C, C 2 ; arrowhead). However, the tumor was demarcated by MADR MAX SM BFP-V5 labeled cells, which had overgrown the hemisphere (Fig. 15C, C3).
• dRMCE-mediated somatic transgenesis in vivo is a highly robust, cost-effective method for creating numerous somatic mosaics using one mouse line.
• dRJVICE has been utilized recently in vitro but suffers from relatively low efficiency of insertions, thus requiring antibiotic selection of clonal cell lines and rigorous validation of individual cell lines with molecular biological methods (PCR, southern blot, etc.).
• PCR molecular biological methods
• episomal and transposon mediated insertion often leads to the supraphysiological expression from tens or hundreds of copies of the given transgene.
• episomal transgenes will more often dilute in proliferating lineages and transpon-based methods will cause cassette "hopping" in the presence of transposases, potentially disrupting endogenous gene function, including tumor suppressors, cell cycle genes, etc.
• the mt/mg reporters to delineate recombinase events, by titrating plasmid constituents and by using multiple donor plasmids, one can track multiple independent lineages.
• Transposons such as PB and Sleeping Beauty
• PB and Sleeping Beauty are increasingly used in combination with EP to produce stable somatic trangenics with several reports utilizing these techniques in recent years.
• Transposons are extremely attractive tools because they allow long-term developmental studies and also in vivo tumor generation.
• Our new method overcomes transposon system's two major problems: random genomic insertions and copy number variability. Notch signaling is one key example of gene-dosage-sensitive molecular pathways. Additionally, several other cell-fate determinants have been shown to result in dramatically differential phenotypes based on their expression levels.
• This strategy can be employed with any off-the-shelf GEMM harboring dual recombinase sites (e.g. Ail4, R26-CAG-LF-mTFPl, Ribotag, and IKMC mice), allowing for the definitive assessment of GOF and LOF protein functions in vivo at a defined dosage (Fig. 16).
• Ail4 is another widely employed reporter mouse line that are typically crossbred with recombinase-expressing transgenic mice.
• donor plasmids can be created for use in Ail4 mice.
• Ribotag use Cre recombination to swap an untagged Rpl22 Exon 4 with an HA-tagged variant for affinity immunoprecipitation of Cre expression- defined mRNA.
• an alternate tag can be inserted with additional elements to allow for simultaneous orthogonal mRNA purification using sequential immunoprecipitation for the tags.
• a donor plasmid with a fluorescent reporter can be simply electroporated (with Flp and Cre) to enable lineage-tracing from a focal point without the need for Cre-expressing mice in various transgenic mice that flank important loci with loxP and FRT sites.
• EP can deliver this large fragment at limiting dilutions into the genome in combination with additional plasmids carrying vCre/sCre-activatable reporters. This type of study would effectively enable GEMM-like, higher-order lineage tracing studies.
• a promoter-less donor vector comprising: a polyadenylation signal or transcription stop element upstream from a transgene or RNA; the transgene or RNA; and paired recombinase recognition sites.
• the promoter-less donor vector further comprises a post-transcriptional regulatory element.
• the promoter-less donor vector further comprises a polyadenylation signal downstream from the transgene or RNA.
• the promoter-less donor vector comprises PGK polyadenylation signal (pA); trimerized SV40pA; a transgene or RNA; loxP; flippase recognition target (FRT); a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); and a rabbit beta-globin pA.
• pA PGK polyadenylation signal
• trimerized SV40pA a transgene or RNA
• loxP flippase recognition target
• FRT flippase recognition target
• WPRE woodchuck hepatitis virus post-transcriptional regulatory element
• rabbit beta-globin pA rabbit beta-globin
• a promoter-less donor vector comprising: a transcription stop element upstream from a transgene or RNA; paired recombinase recognition sites; rtTA-VIO; the transgene or RNA; and TRE-Bi.
• the promoter-less donor vector further comprises a post-transcriptional regulatory element.
• the promoter-less donor vector further comprises a gene encoding puromycin.
• the promoter-less donor vector comprises: a transcription stop element upstream from a transgene or RNA; loxP; rtTA-VIO; the transgene or RNA; TRE-Bi; and flippase recognition target (FRT).
• the promoter-less donor vector further comprises a post-transcriptional regulatory element.
• the promoter-less donor vector further comprises a gene encoding puromycin
• a promoter-less donor vector comprising: a transcription stop element upstream from a transgene or RNA; paired recombinase recognition sites; rtTA-VIO; the transgene or RNA; and TRE-Bi-Dlll .
• the promoter-less donor vector further comprises a post-transcriptional regulatory element.
• the promoter-less donor vector further comprises a gene encoding puromycin.
• the promoter-less donor vector comprises: a transcription stop element upstream from a transgene or RNA; loxP; rtTA-VIO; the transgene or RNA; TRE-Bi-Dlll; and flippase recognition target (FRT).
• the promoter-less donor vector further comprises a gene encoding puromycin.
• the orientation of the paired recombinase sites are dictated by the engineered locus in the transgenic animal (i.e., the recombinase sites will exactly mimic the outer pair of recombinase sites if the transgene is to be inserted in the 'sense' direction of the upstream promoter). Accordingly, in various embodiments, the paired recombinase recognition sites are in the correct orientation. In various embodiments, the loxP is not in an inverted orientation.
• the paired recombinase recognition sites are loxP and flippase recognition target (FRT), and the recombinases are ere and flp.
• Flp include flpE and flpO.
• recombinases include but are not limited to
• the RNA is siRNA, shRNA, or sgRNA.
• the transgene or the RNA comprises disease associated mutations. In various embodiments, the transgene or the RNA comprises a gain- of-function (GOF) mutation. In various embodiments, the transgene or the RNA comprises a loss-of-function (LOF) mutation.
• GAF gain- of-function
• LEF loss-of-function
• transgenes include but are not limited to oncogenic gain-of- function mutations, including Ras, H3f3a, Pdgfra, Trp53 point mutations, Idhl .
• RNAs include but are not limited to tumor suppressor targets such as Trp53, Nfl, Atrx, or Pten.
• post-transcriptional regulatory element examples include but are not limited to Hepatitis B virus post-transcriptional regulatory element (HPRE) and Woodchuck Hepatitis virus post-transcriptional regulatory element (WPRE).
• HPRE Hepatitis B virus post-transcriptional regulatory element
• WPRE Woodchuck Hepatitis virus post-transcriptional regulatory element
• the promoter-less donor vectors described above and below further comprise a gene encoding an antibiotic.
• the promoter-less donor vector further comprises a gene encoding puromycin, or eukaryotic alternatives (kanomycin, blasticidin, etc.).
• the promoter-less donor vector described above and below further comprises a gene encoding a doxycycline-regulated transactivator or CRISPR/Cas based variant (e.g., SpCas9 or Cpfl) for DNA cleavage, gene activation (Crispra), or gene repression (Crispri) when paired with an sgRNA.
• CRISPR/Cas based variant e.g., SpCas9 or Cpfl
• the promoter-less donor vectors described above and below further comprises a gene encoding one or more spaghetti monster fluorescent proteins (SM FPs).
• SM FPs spaghetti monster fluorescent proteins
• the one or more SM FPs are each different SM FPs.
• the one or more SM FPs are four different SM FPs.
• the promoter-less donor vector further comprises a gene encoding one or more epitope tags.
• epitope tags may be the repeats or different tags.
• the epitope tag is an HA tag, Myc tag, V5 tag, or Flag tag.
• the promoter-less donor vector comprises a large- cargo bacterial artificial chromosomes (BAC).
• Various embodiments of the present invention provide for a system for use in in vivo dual recombinase-mediated cassette exchange.
• the system comprises: a promoter-less donor vector as disclosed herein; and one expression vector, comprising two genes encoding recombinases specific to the paired recombinase recognition sites.
• the system comprises: a promoter-less donor vector as disclosed herein; and two expression vectors, the first expression vector comprising a first gene encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second expression vector comprising a second gene encoding a second recombinase that is specific to the other of the paired recombinase recognition sites.
• the system comprises: a promoter-less donor vector, comprising a polyadenylation signal or transcription stop element upstream from a transgene or RNA, the transgene or RNA, and paired recombinase recognition sites; and one expression vector, comprising two genes encoding recombinases specific to the paired recombinase recognition sites.
• the system comprises: a promoter-less donor vector, comprising a polyadenylation signal or transcription stop element upstream from a transgene or RNA, the transgene or RNA, and paired recombinase recognition sites; and two expression vectors, the first expression vector comprising a first gene encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second expression vector comprising a second gene encoding a second recombinase that is specific to the other of the paired recombinase recognition sites.
• the promoter-less donor vector further comprises a post-transcriptional regulatory element. In various embodiments, the promoter-less donor vector further comprises a polyadenylation signal downstream from the transgene or RNA.
• the promoter-less donor vector comprises: PGK polyadenylation signal (pA); trimerized SV40pA; a transgene or RNA; loxP; flippase recognition target (FRT); a rabbit beta-globin pA; and a woodchuck hepatitis virus post- transcriptional regulatory element (WPRE).
• pA PGK polyadenylation signal
• trimerized SV40pA a transgene or RNA
• loxP flippase recognition target
• FRT flippase recognition target
• rabbit beta-globin pA a woodchuck hepatitis virus post- transcriptional regulatory element
• the orientation of the paired recombinase sites are dictated by the engineered locus in the transgenic animal (i.e., the recombinase sites will exactly mimic the outer pair of recombinase sites if the transgene is to be inserted in the 'sense' direction of the upstream promoter). Accordingly, in various embodiments, the paired recombinase recognition sites are in the correct orientation. In various embodiments, the loxP is not in an inverted orientation.
• the paired recombinase recognition sites are loxP and flippase recognition target (FRT), and the recombinases are ere and flp.
• Flp include flpE and flpO.
• recombinases include but are not limited to
• Vlox and derivatives Vlox and derivatives
• SCre Slox and derivatives
• Dre Rox and derivatives
• phiC31 attb
• the RNA is siRNA, shRNA, or sgRNA.
• the transgene or the RNA comprises disease associated mutations. In various embodiments, the transgene or the RNA comprises a gain- of-function (GOF) mutation. In various embodiments, the transgene or the RNA comprises a loss-of-function (LOF) mutation.
• GAF gain- of-function
• LEF loss-of-function
• transgenes include but are not limited to oncogenic gain-of- function mutations, including Ras, H3f3a, Pdgfra, Trp53 point mutations, Idhl .
• RNAs include but are not limited to tumor suppressor targets such as Trp53, Nfl, Atrx, or Pten.
• post-transcriptional regulatory element examples include but are not limited to Hepatitis B virus post-transcriptional regulatory element (HPRE) and Woodchuck Hepatitis virus post-transcriptional regulatory element (WPRE).
• HPRE Hepatitis B virus post-transcriptional regulatory element
• WPRE Woodchuck Hepatitis virus post-transcriptional regulatory element
• the promoter-less donor vectors described above and below further comprise a gene encoding an antibiotic.
• the promoter-less donor vector further comprises a gene encoding puromycin, or eukaryotic alternatives (kanomycin, blasticidin, etc.).
• the promoter-less donor vector described above and below further comprises a gene encoding a doxycycline-regulated transactivator or CRISPR/Cas based variant (e.g., SpCas9 or Cpfl) for DNA cleavage, gene activation (Crispra), or gene repression (Crispri) when paired with an sgRNA.
• CRISPR/Cas based variant e.g., SpCas9 or Cpfl
• the promoter-less donor vectors described above and below further comprises a gene encoding one or more spaghetti monster fluorescent proteins (SM FPs).
• SM FPs spaghetti monster fluorescent proteins
• the one or more SM FPs are each different SM FPs.
• the one or more SM FPs are four different SM FPs.
• the promoter-less donor vector further comprises a gene encoding one or more epitope tags.
• epitope tags may be the repeats or different tags.
• the epitope tag is an HA tag, Myc tag, V5 tag, or Flag tag.
• the promoter-less donor vector comprises a large- cargo bacterial artificial chromosomes (BAC).
• Various embodiments of the present invention provide for a non-human animal model and methods of generating the non-human animal model.
• the animal model can be used to model cancer driver mutations (e.g., gain of function oncogenes).
• generating the non-human animal model comprises providing a system for dual recombinase-mediated cassette exchange; administering the system for dual recombinase-mediated cassette exchange to the non-human animal, and subjecting the non-human animal to electroporation.
• the system for dual recombinase-mediated cassette exchange provided and used to generate the non-human animal model is a system for dual recombinase-mediated cassette exchange of present invention as described herein.
• the method further comprises administering hypBase and/or reporter plasmids.
• the system for dual recombinase-mediated cassette exchange electroporation can be administered to a location wherein testing is desired.
• the system can be injected into the lateral ventricle and electroporation can be conducted at that location.
• the system can be injected, for example, into the cerebral spinal fluid, or into or near the spinal cord, and electroporation can done at or near the spinal cord.
• the non-human animal model comprises a system for dual recombinase-mediated cassette exchange as disclosed herein, wherein the dual Recombinase-Mediated Cassette Exchange has occurred.
• the non-human animal is a mouse.
• the non-human animal is a rat, hamster, gerbil, pig, guinea pig, rabbit, monkey (e.g., rhesus monkey), baboon, chimpanzee, sheep, or dog.
• the non- human animal is a genetically engineered mouse.
• the non-human animal is a genetically engineered mouse with paired dual recombinase sites.
• the mouse is a Rosa26mTmG (mTmG), Ail4, R26-CAG-LF-mTFPl, or IKMC mouse or mouse with similar paired dual recombinase sites.
• Various embodiments of the present invention provide for a method of screening a drug candidate, comprising: providing a non-human animal model of the present invention, administering the drug candidate, and assessing the effects of the drug candidate on the non-human animal model.
• the method further comprises selecting the drug candidate as a drug when the drug candidate provides a beneficial result to the non-human animal model. In various embodiments, the method further comprises validating the drug candidate as a drug when the drug candidate provides a beneficial result to the non-human animal model.
• “Beneficial result” may include, but are in no way limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition and prolonging a patient's life or life expectancy.
• Various embodiments of the present invention provide a method of treating a disease or condition in a subject, comprising: providing a cell comprising a system for dual recombinase-mediated cassette exchange of the present invention and administering the cell to the subj ect.
• the cell is a stem cell.
• dual Recombinase-Mediated Cassette Exchange has occurred in the cell.
• Administering the cell to the subject can be performed by a route of administration that is appropriate for the disease condition.
• Route of administration may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal or parenteral.
• Transdermal administration may be accomplished using a topical cream or ointment or by means of a transdermal patch.
• Parenteral refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.
• the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders.
• the pharmaceutical compositions can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release.
• the compositions may be in the form of solutions or suspensions for infusion or for injection.
• the pharmaceutical compositions based on compounds according to the invention may be formulated for treating the skin and mucous membranes and are in the form of ointments, creams, milks, salves, powders, impregnated pads, solutions, gels, sprays, lotions or suspensions. They can also be in the form of microspheres or nanospheres or lipid vesicles or polymer vesicles or polymer patches and hydrogels allowing controlled release.
• These topical-route compositions can be either in anhydrous form or in aqueous form depending on the clinical indication.
• the cell is administered by transplantation.
• Treatments include using transplanted cells with the dRMCE event to deliver growth factors, exosomal, or peptide therapies in diseases such as cancer, neurodegenerative disease, stroke, or epilepsy.
• the cell is transplanted and then subsequently targeted for dRMCE by electroporation or viral transduction.
• Flp-Cre constructs were generously provided by Y. Voziyanov and previously validated in the context of dRMCE.
• Our donor plasmids were derived from PGKneotpAlox2, using In-Fusion technique (Clontech) in combination with standard restriction digestion techniques. FRT site was created by annealing two oligos and infusing the insert into PGKneotpAlox2. Downstream generation of other donor plasmids were done by removing the existing ORF and adding a new cassette using In-Fusion or ligation.
• PB-CAG-plasmids were previously described.
• Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J mice were purchased from the Jackson Laboratory. mTmG were bred with wildtype CD1 mice (Charles River) to generate heterozygous mice. All mice were used in accordance with the Cedars-Sinai Institutional Animal Care and Use Committee. Postnatal lateral ventricle EPs were performed as previously described. PI -3 pups were placed on ice for ⁇ 5 min. All DNA mixtures contained 0.5-1 ⁇ g/ ⁇ l of Flp-Cre expression vector, donor plasmid, hypBase, or CAG-reporter plasmids diluted in Tris-EDTA buffer unless noted otherwise.
• mice brains were isolated and fixed in 4% PFA overnight at
• Brains were embedded in 4% low melting point agarose and sectioned at 70 ⁇ on a vibratome (Leica).
• Immunohistochemistry was performed using standard methodology as previously described. All secondary antibodies (Jackson ImmunoResearch) were used at 1 :500. Details on the primary antibodies can be found in Table 3.
• Doxycycline (Clontech 63131 1) was added to culture media at the final concentration of lOOng/ml.
• Puromycin (Clontech 631305) was used at ⁇ g/ml.
• FlEx-based transgene expression specifically Cre- mediated inversion and activation of EGFP cassette (FlEx-EGFP).
• FlEx-EGFP EGFP cassette
• FlEx-multi-miR-E CAG-driven FlEx-based construct harboring the multiple miR-Es
• Postnatal mNSC line was established by dissociating CD1 pup brains, transfected with EGFP or FlEx-multi-miR-E and Cre-recombinase vector. Fluorescent cells were sorted and subjected to mRNA extraction and SYBR-based Fluidigm BioMark dynamic array using qPCR probes for Nfl, Pten, and Trp53.
• a short reverse primer and an ultramer forward primer were combined in a PCR reaction and subsequent purification to make concentrated sgRNAs (Ran et al, 2013). lOOng of each fragment was combined with plasmid DNA for EP.
• a pure population of tumor cells was obtained by FACS and genomic DNA was isolated (Qiagen DNeasy). Using primers flanking the gRNA target site, we PCR amplified the regions expected to contain InDel mutations for NF1, Trp53, and Pten. The PCR amplified fragments were topo cloned using the Thermo Fisher Zero Blunt TOPO kit and transformed into One Shot MAX Efficiency DH5-T1R cells.

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