loxP Sites Research Articles

Generation of floxed Spag6l mice and disruption of the gene by crossing to a Hprt-Cre line.

Mouse sperm-associated antigen 6 like (SPAG6L) is an axoneme central apparatus protein, essential for the normal function of the ependymal cell and lung cilia, and sperm flagella. Accumulated evidence has disclosed multiple biological functions of SPAG6L, including ciliary/flagellar biogenesis and polarization, neurogenesis, and neuronal migration. Conventional Spag6l knockout mice died of hydrocephalus, which impedes further investigation of the function of the gene in vivo. To overcome the limitation of the short lifespan of conventional knockout mice, we developed a conditional allele by inserting two loxP sites in the genome flanking exon 3 of the Spag6l gene. By crossing the floxed Spag6l mice to a Hrpt-Cre line which expresses Cre recombinase ubiquitously in vivo, mutant mice that are missing SPAG6L globally were obtained. Homozygous mutant Spag6l mice showed normal appearance within the first week after birth, but reduced body size was observed after 1week, and all developed hydrocephalus and died within 4 weeks of age. The phenotype mirrored that of the conventional Spag6l knockout mice. The newly established floxed Spag6l model provides a powerful tool to further investigate the role of the Spag6l gene in individual cell types and tissues.

A Dual-Function "TRE-Lox" System for Genetic Deletion or Reversible, Titratable, and Near-Complete Downregulation of Cathepsin D.

Commonly employed methods for reversibly disrupting gene expression, such as those based on RNAi or CRISPRi, are rarely capable of achieving >80-90% downregulation, making them unsuitable for targeting genes that require more complete disruption to elicit a phenotype. Genetic deletion, on the other hand, while enabling complete disruption of target genes, often produces undesirable irreversible consequences such as cytotoxicity or cell death. Here we describe the design, development, and detailed characterization of a dual-function "TRE-Lox" system for effecting either (a) doxycycline (Dox)-mediated downregulation or (b) genetic deletion of a target gene-the lysosomal aspartyl protease cathepsin D (CatD)-based on targeted insertion of a tetracycline-response element (TRE) and two LoxP sites into the 5' end of the endogenous CatD gene (CTSD). Using an optimized reverse-tetracycline transrepressor (rtTR) variant fused with the Krüppel-associated box (KRAB) domain, we show that CatD expression can be disrupted by as much as 98% in mouse embryonic fibroblasts (MEFs). This system is highly sensitive to Dox (IC50 = 1.46 ng/mL) and results in rapid (t1/2 = 0.57 d) and titratable downregulation of CatD. Notably, even near-total disruption of CatD expression was completely reversed by withdrawal of Dox. As expected, transient expression of Cre recombinase results in complete deletion of the CTSD gene. The dual functionality of this novel system will facilitate future studies of the involvement of CatD in various diseases, particularly those attributable to partial loss of CatD function. In addition, the TRE-Lox approach should be applicable to the regulation of other target genes requiring more complete disruption than can be achieved by traditional methods.

Deletion of LCMR1 in alveolar type II cells induces lethal impairment of lung structure and function in adult mice.

Previous studies of lung cancer metastasis-related protein 1 (LCMR1) mainly focused on its relationship with cancer. However, the function of LCMR1 in normal tissues or cells is poorly understood. We aimed to investigate the effects of alveolar type II cell (AT2 cell)-specific LCMR1 deletion on lung structure and function in adult mice. Mice carrying the floxed LCMR1 allele with exons 2-4 flanked by loxP sites were constructed and then crossed with Sftpc-CreERT2 mice to obtain Sftpc-CreERT2 ; LCMR1 flox/flox for AT2 cell-specific LCMR1 deletion and LCMR1 flox/flox mice as littermate control. We observed the body weight change, histopathology, lung wet/dry weight ratio, pulmonary function, and survival of the mice, together with the protein concentration, inflammatory cells, and cytokine levels in bronchoalveolar lavage fluid. We also detected AT2 cell numbers and expression of pulmonary surfactant protein in the lung tissues. The apoptosis of AT2 cells was also assessed. We found that AT2 cell-specific LCMR1 deletion caused rapid weight loss and increased mortality in mice. Histopathological analysis revealed damaged lung structure, including inflammatory cell infiltration, alveolar hemorrhage, and edema. The lung wet/dry weight ratio was higher and bronchoalveolar lavage fluid (BALF) analysis revealed elevated protein concentration, inflammatory cell counts, and cytokine levels. Pulmonary function measurement showed increased airway resistance, decreased lung compacity, and compliance. We also found massive AT2 cell loss and altered expression of pulmonary surfactant protein. Deletion of LCMR1 promoted apoptosis in AT2 cells. We successfully generated an AT2 cell-specific LCMR1 conditional knockout mouse model and further revealed the crucial role of LCMR1 in maintaining AT2 cell homeostasis.

DEVELOPMENT OF MURINE STEM CELLS WITH CONDITIONAL KNOCKOUT OF HUMANIZED SNCA GENE

α-synuclein is one of the key molecular links in the pathogenesis of Parkinson’s disease. The accumulated data indicate that pathogenic mutations in the Snca gene are associated with the development of neurodegenerative brain damage, indicating the relevance of studying the synuclein neurobiological role.The aim of the study was to create a genetically modified clone of mouse stem cells with a conditional knockout of humanized α-synuclein, which can be used for the reinjection into mouse blastocysts, as well as for basic and applied in vitro research in the field of pathophysiology and neuropharmacology.Materials and methods. To create mouse stem cells with a conditional knockout of the humanized Snca gene, a previously obtained clone with the first Snca exon flanked by LoxP sites, was used. The CRISPR/Cas9-mediated homologous recombination system with donor DNA oligonucleotides of the human sites of the corresponding gene sites was used to humanize the fourth and fifth exons. Cas9 nuclease, single guide RNA, and donor DNA were transfected into mouse cells.Results. An approach to obtaining clones of mouse genetically modified stem cells expressing pathological humanized α-synuclein, has been proposed and implemented. The resulting clones were plated on Petri dishes for propagation and a further genetic analysis. Clone 126-2F4 was found out carrying the necessary genetic modifications. The results obtained are fundamentally important not only for understanding the development of the pathological process in α-synucleinopathies, but which is more important, for the development of new therapeutic approaches that will stop the extension of the human α-synuclein aggregation pathology throughout the nervous system, and the validation of these approaches in preclinical trials.Conclusion. As a result of the study, a strategy for CRISPR/Cas9-assisted homologous recombination in the genome of mouse embryonic stem cells has been developed to create a fully humanized Snca gene encoding α-synuclein, and the clone genome of mouse embryonic stem cells has been edited using a CRISPR technology. The RNA and DNA oligonucleotides necessary for the creation of RNP complexes that carry out a directed homologous recombination in the Snca locus of the mouse genome have been synthesized. The developed cell clone can serve to create a line of genetically modified mice that serve as a test system for pathophysiological and neuropharmacological studies associated with synucleinopathies. Herewith, before the induction of the Cre-dependent recombination, this line is a representative model for studying a biological role of mutant Snca. At the same time, after a Cre-dependent knockout activation, it is possible to imitate the pharmacological inhibition of α-synuclein, which is of particular interest for applied research in neuropharmacology.

Generation of Conditional Knockout Alleles for PRUNE-1.

PRUNE1 is a member of the aspartic acid-histidine-histidine (DHH) protein superfamily, which could display an exopolyphosphatase activity and interact with multiple cellular proteins involved in the cytoskeletal rearrangement. It is widely expressed during embryonic development and is essential for embryogenesis. PRUNE1 could also be critical for postnatal development of the nervous system as it was found to be mutated in patients with microcephaly, brain malformations, and neurodegeneration. To determine the cellular function of PRUNE1 during development and in disease, we have generated conditional mouse alleles of the Prune1 in which loxP sites flank exon 6. Crossing these alleles with a ubiquitous Cre transgenic line resulted in a complete loss of PRUNE1 expression and embryonic defects identical to those previously described for Prune1 null embryos. In addition, breeding these alleles with a Purkinje cell-specific Cre line (Pcp2-Cre) resulted in the loss of Purkinje cells similar to that observed in patients carrying a mutation with loss of PRUNE1 function. Therefore, the Prune1 conditional mouse alleles generated in this study provide important genetic tools not only for dissecting the spatial and temporal roles of PRUNE1 during development but also for understanding the pathogenic role of PRUNE1 dysfunction in neurodegenerative or neurodevelopmental disease. In addition, from this work, we have described an approach that allows one to efficiently generate conditional mouse alleles based on mouse zygote electroporation.

Antibody-based protection against respiratory syncytial virus in mice and their offspring through vectored immunoprophylaxis.

Respiratory syncytial virus (RSV) causes acute lower respiratory tract infections, with potential lower respiratory tract infections, which can be particularly problematic in infants and the elderly. There are no approved vaccines for RSV. The current standard of care for high-risk individuals is monthly administration of palivizumab, a humanized murine monoclonal antibody (mAb) targeting the RSV fusion protein. Adeno-associated virus (AAV)-mediated expression of mAbs has previously led to sustained expression of therapeutic concentrations of mAbs in several animal models, representing an alternative to repetitive passive administration. Intramuscular (IM) administration of AAV6.2FF expressing RSV antibodies, palivizumab or hRSV90, resulted in high concentrations of human (h)IgG1 mAbs in the serum and at various mucosal surfaces, while intranasal administration limited hIgG expression to the respiratory tract. IM administration of AAV6.2FF-hRSV90 or AAV6.2FF-palivizumab in a murine model provided sterilizing immunity against challenge with RSV A2. Evidence of maternal passive transfer of vectorized hRSV90 was detected in both murine and ovine models, with circulating mAbs providing sterilizing immunity in mouse progeny. Finally, addition of a "kill switch" comprised of LoxP sites flanking the mAb genes resulted in diminished serum hIgG after AAV-DJ-mediated delivery of Cre recombinase to the same muscle group that was originally transduced with the AAV-mAb vector. The ability of this AAV-mAb system to mediate robust, sustained mAb expression for maternal transfer to progeny in murine and ovine models emphasizes the potential of this platform for use as an alternative prophylactic vaccine for protection against neonatal infections, particularly in high-risk infants.

Conformational selection in the Cre CBD domain: Insights into intasome assembly.

During site-specific recombination tyrosine recombinases promote scission, insertion, and recombination of specific DNA sequences. The Cre/loxP system from P1 bacteriophage promotes viral DNA circularization after infection and equitable plasmid assortment during cell division. It acts through the formation of a tetramer (intasome) in which Cre monomers are bound in a head-to-tail fashion to a half loxP site, alternating between active and inactive conformations. Despite knowledge of the recombination mechanism and strand exchange order, information regarding target selection and intasome assembly is still missing. We used molecular dynamics simulations (MD) to assess the conformational flexibility of the CBD domain of Cre recombinase. The starting seeds for MD were structures with PDB ID 1q3u (an intasome) and 7rhy (a Cre monomer bound to a loxP half-site); these structures vary in the position of helix A. We simulated four replicas for each system for 5 microseconds in an explicit solvent model (TIP3P) with the software GROMACS using CHARMM36m at 300K and 1 atm. Van der Waals interactions were calculated using a 1.2 nm cutoff and PME for electrostatics, with a 4 fs timestep. Our results show that helix A can switch from an intasome-like to a monomer-like conformation, passing through distinct intermediate and unfolded states. These results suggest that the position of helix A is selected concomitantly with establishing monomer-monomer interactions during intasome assembly. Also, the flexibility of helix A could help to interpret NMR data in which the signals for this helix are missing.We acknowledge the computer resources provided by the LNS del Sureste de México through grant 202101023N, Laboratorio de Supercómputo y Visualización en Paralelo, and LANCAD through grants 99-2021 and 49-2022, and CONACyT (graduate school scholarship 858905, grant INF-2014-02-231504, grant A1-S-11842).

Liver-restricted deletion of the biliary atresia candidate gene Pkd1l1 causes bile duct dysmorphogenesis and ciliopathy.

A recent multicenter genetic exploration of the biliary atresia splenic malformation syndrome identified mutations in the ciliary gene PKD1L1 as candidate etiologic contributors. We hypothesized that deletion of Pkd1l1 in developing hepatoblasts would lead to cholangiopathy in mice. CRISPR-based genome editing inserted loxP sites flanking exon 8 of the murine Pkd1l1 gene. Pkd1l1Fl/Fl cross-bred with alpha-fetoprotein-Cre expressing mice to generate a liver-specific intrahepatic Pkd1l1 -deficient model (LKO). From embryonic day 18 through week 30, control ( Fl/Fl ) and LKO mice were evaluated with standard serum chemistries and liver histology. At select ages, tissues were analyzed using RNA sequencing, immunofluorescence, and electron microscopy with a focus on biliary structures, peribiliary inflammation, and fibrosis. Bile duct ligation for 5 days of Fl/Fl and LKO mice was followed by standard serum and liver analytics. Histological analyses from perinatal ages revealed delayed biliary maturation and reduced primary cilia, with progressive cholangiocyte proliferation, peribiliary fibroinflammation, and arterial hypertrophy evident in 7- to 16-week-old LKO versus Fl/Fl livers. Following bile duct ligation, cholangiocyte proliferation, peribiliary fibroinflammation, and necrosis were increased in LKO compared with Fl/Fl livers. Bile duct ligation of the Pkd1l1 -deficient mouse model mirrors several aspects of the intrahepatic pathophysiology of biliary atresia in humans including bile duct dysmorphogenesis, peribiliary fibroinflammation, hepatic arteriopathy, and ciliopathy. This first genetically linked model of biliary atresia, the Pkd1l1 LKO mouse, may allow researchers a means to develop a deeper understanding of the pathophysiology of this serious and perplexing disorder, including the opportunity to identify rational therapeutic targets.

Fluorescent PCR-based Screening Methods for Precise Knock-in of Small DNA Fragments and Point Mutations in Zebrafish.

Generation of zebrafish (Danio rerio) models with targeted insertion of epitope tags and point mutations is highly desirable for functional genomics and disease modeling studies. Currently, CRISPR/Cas9-mediated knock-in is the method of choice for insertion of exogeneous sequences by providing a repair template for homology-directed repair (HDR). A major hurdle in generating knock-in models is the labor and cost involved in screening of injected fish to identify the precise knock-in events due to low efficiency of the HDR pathway in zebrafish. Thus, we developed fluorescent PCR-based high-throughput screening methods for precise knock-in of epitope tags and point mutations in zebrafish. Here, we provide a step-by-step guide that describes selection of an active sgRNA near the intended knock-in site, design of single-stranded oligonucleotide (ssODN) templates for HDR, quick validation of somatic knock-in using injected embryos, and screening for germline transmission of precise knock-in events to establish stable lines. Our screening method relies on the size-based separation of all fragments in an amplicon by fluorescent PCR and capillary electrophoresis, thus providing a robust and cost-effective strategy. Although we present the use of this protocol for insertion of epitope tags and point mutations, it can be used for insertion of any small DNA fragments (e.g., LoxP sites, in-frame codons). Furthermore, the screening strategy described here can be used to screen for precise knock-in of small DNA sequences in any model system, as PCR amplification of the target region is its only requirement. Key features This protocol expands the use of fluorescent PCR and CRISPR-STAT for screening of precise knock-in of small insertions and point mutations in zebrafish. Allows validation of selected sgRNA and HDR template within two weeks by somatic knock-in screening. Allows robust screening of point mutations by combining restriction digest with CRISPR-STAT. Graphical overview Overview of the three-phase knock-in pipeline in zebrafish (created with BioRender.com).

Generation of Floxed Mice by Sequential Electroporation.

Generation of conditional knockout mice using the Cre-loxP system is essential for the analysis of gene functions. The use of CRISPR-Cas9 in combination with two sets of guide RNAs and single-stranded oligonucleotides including loxP sites enables simultaneous insertion of two loxP sequences. Unfortunately, this method induces double-strand breaks at two sites in the same chromosome, which causes an undesirable large chromosomal deletion and reduces the flanked loxP (flox) rate. To overcome this problem, we have developed a method that sequentially introduces each loxP sequence by electroporation at the one- and two-cell embryonic stages, respectively. This sequential electroporation method improves the floxing efficiency compared with the conventional simultaneous method, leading to a high yield of offspring with floxed alleles.

One-step generation of a conditional allele in mice using a short artificial intron

Despite tremendous advances in genome editing technologies, generation of conditional alleles in mice has remained challenging. Recent studies in cells have successfully made use of short artificial introns to engineer conditional alleles. The approach consists of inserting a small cassette within an exon of a gene using CRISPR-Cas9 technology. The cassette, referred to as Artificial Intron version 4 (AIv4), contains sequences encoding a splice donor, essential intronic sequences flanked by loxP sites and a splice acceptor site. Under normal conditions, the artificial intron is removed by the splicing machinery, allowing for proper expression of the gene product. Following Cre-mediated recombination of the two loxP sites, the intron is disabled, and splicing can no longer occur. The remaining intronic sequences create a frameshift and early translation termination. Here we describe the application of this technology to engineer a conditional allele in mice using Scyl1 as a model gene. Insertion of the cassette occurred in 17% of edited mice obtained from pronuclear stage zygote microinjection. Mice homozygous for the insertion expressed SCYL1 at levels comparable to wild-type mice and showed no overt abnormalities associated with the loss of Scyl1 function, indicating the proper removal of the artificial intron. Inactivation of the cassette via Cre-mediated recombination in vivo occurred at high frequency, abrogated SCYL1 protein expression, and resulted in loss-of-function phenotypes. Our results broaden the applicability of this approach to engineering conditional alleles in mice.

A novel genetic approach to specifically target and manipulate plaque‐associated microglia in mice

AbstractBackgroundWithin the Alzheimer’s disease (AD) brain, microglia surround extracellular plaques and mount a chronic inflammatory response. These plaque‐associated microglia (PAMs) are characterized by specific disease‐associated gene expression signatures, including Apoe, Cst7, Trem2, Clec7a, Itgax, and Csf1. Data from animal models of AD have identified that PAMs play critical roles in plaque formation and plaque compaction, but also contribute to synaptic and neuronal dysfunction and loss. To better understand the role of microglia in AD, we need precise models which directly target specific subsets of microglia, however; no such model currently exists. Here, we developed a novel genetic cre‐recombinase approach to specifically target and manipulate PAMs in AD mouse models. We identified that of PAM‐identified genes Cst7 shows no expression in the homeostatic mouse brain and is specifically and highly expressed in PAMs.MethodTo avoid caveats associated with tamoxifen inducible cre recombinase systems, we instead established a destabilized cre‐recombinase (ddCre) mouse line, in which Cre becomes stabilized and facilitates recombination in the presence of the antibiotic trimethoprim. ddCre was knocked‐in to the Cst7 locus (Cst7ddCre), followed by a P2A linker to ensure normal expression of Cst7 transcripts. We crossed these mice to a 5xFAD mouse model of AD and a loxp‐stop‐loxp‐tdTomato (Ai14tdtomato) reporter line. Upon the administration trimethoprim, cre‐recombination at loxp sites in Cst7 expressing cells occurs and tdTomato is expressed.ResultWe found that 5xFAD/Cst7 ddCre/Ai14tdtomato mice showed specific tdTomato fluorescence in PAMs with no expression present in non‐plaque associated microglia, or other cell types throughout the brain.ConclusionAltogether, we have shown that our novel Cst7 ddCre mouse line effectively targets PAMs. With this validation, we can alter any single gene specifically in PAMs.

186 Dissecting the role of the common neurotrophin receptor CD271 in the skin: generation and characterization of a novel mouse model with keratinocyte-specific conditional deletion

The common neurotrophin receptor CD271, also called p75NTR, is the transmembrane low-affinity receptor for neurotrophins, belonging to the TNF superfamily. In the skin, CD271 is involved in the differentiation process and seems like to be implicated in cutaneous pathogenesis. To date, different animal models have been developed to obtain null mutations of CD271, but they defy to explain CD271 precise role in keratinocytes. By using a Keratin14-Cre mediated excision of the CD271 gene flanked by loxP sites, we generated a novel mouse model with a conditional deletion of CD271 in the epidermis.

Gata3 ZsG and Gata3 ZsG-fl: Novel murine Gata3 reporter alleles for identifying and studying Th2 cells and ILC2s in vivo.

T helper-2 (Th2) cells and type 2 innate lymphoid cells (ILC2s) play crucial roles during type 2 immune responses; the transcription factor GATA3 is essential for the differentiation and functions of these cell types. It has been demonstrated that GATA3 is critical for maintaining Th2 and ILC2 phenotype in vitro; GATA3 not only positively regulates type 2 lymphocyte-associated genes, it also negatively regulates many genes associated with other lineages. However, such functions cannot be easily verified in vivo because the expression of the markers for identifying Th2 and ILC2s depends on GATA3. Thus, whether Th2 cells and ILC2s disappear after Gata3 deletion or these Gata3-deleted "Th2 cells" or "ILC2s" acquire an alternative lineage fate is unknown. In this study, we generated novel GATA3 reporter mouse strains carrying the Gata3 ZsG or Gata3 ZsG-fl allele. This was achieved by inserting a ZsGreen-T2A cassette at the translation initiation site of either the wild type Gata3 allele or the modified Gata3 allele which carries two loxP sites flanking the exon 4. ZsGreen faithfully reflected the endogenous GATA3 protein expression in Th2 cells and ILC2s both in vitro and in vivo. These reporter mice also allowed us to visualize Th2 cells and ILC2s in vivo. An inducible Gata3 deletion system was created by crossing Gata3 ZsG-fl/fl mice with a tamoxifen-inducible Cre. Continuous expression of ZsGreen even after the Gata3 exon 4 deletion was noted, which allows us to isolate and monitor GATA3-deficient "Th2" cells and "ILC2s" during in vivo immune responses. Our results not only indicated that functional GATA3 is dispensable for regulating its own expression in mature type 2 lymphocytes, but also revealed that GATA3-deficient "ILC2s" might be much more stable in vivo than in vitro. Overall, the generation of these novel GATA3 reporters will provide valuable research tools to the scientific community in investigating type 2 immune responses in vivo.

Developmental Stage-Specific Hematopoietic Failure In Vivo in a Novel Model of Diamond Blackfan Anemia

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome, characterized by failure of erythropoiesis at the progenitor stages, congenital anomalies, and predisposition to cancer. DBA is most frequently due to mutations in genes encoding a ribosomal protein, the most common one being mutations in RPS19. Despite the well-established role of RPS19 as well as other ribosomal proteins in the erythropoietic failure in in vitro culture systems, there is a paucity of in vivo murine models of DBA due to differences between humans and mice. Indeed, stress erythropoiesis occurs in the spleen of mouse, compensating for the erythropoietic defects observed in the bone marrow. Using CRISPR/Cas9 technologies, we generated a conditional knockout of Rps19 by flanking exon 2 with 2 loxP sites and studied hematopoiesis in this model. While Tie2-cre+;Rps19+/- animals die in utero, Vav-iCre+; Rps19+/- mice are born at expected Mendelian ratios with severe macrocytic anemia. Surprisingly, and unlike any other model generated to date, these mice present with reticulocytopenia, a critical characteristic of patients with DBA and die by post-natal day 10 (P10), due to bone marrow failure and decreased splenic stress erythropoiesis. To understand the role of ribosomal proteins during hematopoietic development, we analyzed fetal livers of Vav-iCre+; Rps19+/- embryos and control littermates at E13.5 and E17.5. Using flow cytometry, we observed defective erythropoiesis and gradual hematopoietic stem cell exhaustion at the fetal to neonatal transition. Mechanistically, and consistent with previous studies using in vitro systems, Rps19mutant cells had increased p53 protein expression and reduced mTORC1 activity. Unexpectedly, hematopoietic progenitors had enhanced global protein synthesis. Using BrDU and EdU stainings, we observed a decrease in the cell cycle duration of erythroblasts, consistent with the macrocytosis and reticulocytopenia seen in neonatal Vav-iCre+; Rps19+/- mice. Since p53 is reported to have a central role in the pathophysiology of DBA, we asked whether deletion of one allele of p53 could rescue the defects observed in Vav-iCre+; Rps19+/-. The Vav-iCre+; Rps19+/-, trp53+/-presented with improved blood counts, increased spleen size and cellularity but died by four weeks after birth. Analyses of the hematopoietic compartment in these animals at P21 revealed bone marrow failure and HSC depletion with skewed progenitor populations. The increase in global protein synthesis observed in Vav-iCre+; Rps19+/- remained unchanged in Vav-iCre+; Rps19+/-, trp53+/-, suggesting mechanisms independent of p53. Taken together, we have developed a novel in vivo model for the study of DBA. Our findings could potentially explain why DBA typically manifests after birth and highlight a critical role for Rps19 during hematopoietic differentiation both in utero and after birth.

CBFB-MYH11 Is Required for the Maintenance of Inv(16) AML

Acute myeloid leukemia (AML) is a heterogenous disease arising from the immature blood cells in the bone marrow. One of the most common chromosomal aberrations, inversion of chromosome 16 [inv(16)], generates the fusion gene Cbfb-MYH11 (CM) which is known to initiate leukemogenesis. Because the fusion protein is invariably found in patients at relapse, it is assumed that CM is also required after leukemic transformation. However, knockdown of CM in the inv(16) immortalized cell line ME-1, caused relatively subtle effects on differentiation but did not affect viability. This raises the possibility that CM may be dispensable after leukemic transformation. To address this possibility in primary leukemia cells, we generated a new knock-in mouse model with the CM fusion gene flanked by loxP sites (CbfbflMYH11). This allows for the fusion gene to be deleted by Cre Recombinase (Cre) after leukemia development. To test the effect of loss of CM in vitro, we transduced Cbfb+/flMYH11 leukemia cells from three independent CbfbflMYH11 mice with a lentivirus expressing Cre. We confirmed the deletion of CM by qPCR and western blot. In Cbfb+/flMYH11, Cre+ cells, we observed a statistically significant increase in Annexin V staining and a decrease in colony forming ability as compared to control Cbfb+/flMYH11 cells. To determine if the colonies that did grow from Cre-infected cells had successfully deleted CM, individual colonies were picked. We found that majority of the colonies retained the unexcised CM allele, but 5-20% of colonies had successfully deleted CM, indicating that a rare population of leukemia cells can survive without CMin vitro. To test the requirement for CM in vivo, we used an inducible shRNA against CM rather than Cre+CbfbflMYH11 leukemia cells, as the inducible knockdown model will allow us to avoid potential defects in engraftment. We transduced CM expressing leukemic cells with a lentiviral construct with a doxycycline (Dox) inducible shRNA against MYH11 (shMYH11) that also expresses GFP. To test the feasibility of this approach in vitro, the sorted shCM+ leukemia cells were incubated with Dox for 48 hours. We found that Cbfb-MYH11 mRNA and protein were decreased in Dox treated leukemia cells, as compared to control cells. In addition, Dox treated shCM+ cells showed increased apoptosis and decreased colony forming ability, similar to Cbfb+/flMYH11Cre+ cells. To test the effect of CM knockdown in vivo, shCM transduced leukemia cells were transplanted into sub-lethally irradiated mice and monitored for leukemic engraftment. Once significant leukemic burden in the peripheral blood was detected, mice were given Dox or vehicle in their drinking water. Starting on day 7, we observed a statistically significant decrease in leukemia burden in Dox treated mice in the peripheral blood and spleen, with the percent of leukemia cells below 5% starting on day 14. In the bone marrow, there was a statistically significant decrease in leukemic burden starting on day 14, but the leukemia cells remained at 30-40% through day 28. Our initial tests indicate that the remaining GFP+ leukemia cells in the bone marrow express reduced levels of CM, implying that these cells may not strictly require the fusion protein or may be able to survive with only low levels of Cbfb-MYH11 activity. In a separate, small cohort of shCM+ transplanted mice, we treated the mice with Dox or control water and monitored for leukemia development. All the control treated mice developed fatal leukemia by day 21, whereas the majority of the Dox treated mice survived until day 42 of treatment. Interestingly, among the 6 Dox treated mice, 2 showed re-emergence of GFP+ leukemia cells in the peripheral blood on day 21 and 28, respectively. We are currently testing whether these relapsed leukemia cells re-express CM. Taken together, our results indicate that Cbfb-MYH11 is largely required for leukemia maintenance, although a subset of cells may be able to survive without the fusion gene. These results imply that drugs targeting the Cbfb-MYH11 have the potential to be effective treatments for inv(16) AML, although substantial inhibition of the fusion protein will likely be needed for effective treatment of the disease.

Lnp-Targeting Hematopoietic Stem Cells and Lentiviral Gene Transfer to Generate and Rescue a Novel Mouse Model of Lethal Alpha-Thalassemia

α-Thalassemia (α-thal) is caused by insufficient production of the α-globin protein. Clinical presentation of α-thal varies from an asymptomatic condition (one inactivated α-globin gene) to Bart's Hydrops Fetalis Syndrome/BHFS (complete knockout). In patients with severe α-thal, allogeneic bone marrow transplantation may be required for survival. Alternatively, patients could be treated through autologous BMT following ex vivo transduction with a human α-globin expressing lentiviral vector. However, a proper model system and vector must be established for the development of such a treatment. We crossed animals that had deletion of both Hba-a1 and Hba-a2 in heterozygosity (Hba-a1+/ko/Hba-a2+/ko) to generate knockout (KO) embryonic mice lacking all the α-globin genes (Hba-a1ko/ko/Hba-a2ko/ko). Although these embryos die perinatally, their fetal liver cells (FLC) at 13.5 dpc are still viable and represent a source of engraftable hematopoietic stem cells (HSC). KO-FLC were transplanted into congenic myeloablated WT mice to generate mice carrying Hba-a1ko/ko/Hba-a2ko/ko HSC. These animals developed a severe phenotype ~7 weeks post engraftment. As these mice lack α-globin expression, we observed β-tetramer formation (β4, HbH, Fig1A), resulting in aberrant red blood cell (RBC) morphology, β-globin precipitates, high RBC oxygen binding affinity, elevated erythropoietin, hematocrit and hemoglobin levels, splenomegaly and vaso-occlusive episodes, due to the large number of abnormal RBCs. Furthermore, we observed iron deposition in the liver and kidney, in agreement with very low levels of hepcidin expression in the liver. However, FLC engraftment is cumbersome and requires extensive breeding and many animals to generate enough FLC for few recipients. To facilitate the generation of α-thal mice, we created conditional knockout (cKO) mice that have been edited to remove Hba-a2, while inserting loxP sites flanking Hba-a1 (Hba-a1fl/Hba-a2ko). Homozygous cKO animals (Hba-a1fl/fl/Hba-a2ko/ko) demonstrate a mild phenotype. To achieve complete deletion of the α-globin genes (Hba-a1ko/ko/Hba-a2ko/ko) in HSC, we used lipid nanoparticles (LNPs), which are one of the most promising delivery systems to package and make mRNA into a useful therapeutic. We developed a novel LNP preparation with an antibody to target CD117 and deliver nucleoside-modified mRNA to HSC, (see abstract Breda et al). We treated HSC from homozygous cKO animals (Hba-a1fl/fl/Hba-a2ko/ko) ex vivo with CD117-LNP embedded with Cre mRNAs (Cre-CD117-LNP). Cells showed the expected deletion of the α-globin genes and were injected into myeloablated recipient mice. These chimeras recapitulated the phenotype observed following transplantation of constitutive α-globin-KO FLC, including elevated hematocrit, splenomegaly, and culminating in lethality ~7 weeks post-transplant. We screened multiple potential erythroid specific lentiviral vectors in both mouse and human cell lines and identified a promising candidate, ALS20αI, which at VCN=1 produced human α-globin protein equivalent to that of a single endogenous α-globin gene. Myeloablated recipient mice transplanted with Cre-CD117-LNP-treated cKO BM die roughly 7 weeks post-transplant with the expected pathological phenotype (Fig1A). In contrast, mice receiving BM treated with Cre-CD117-LNP and ALS20αI (VCN>1) all survived long term (>4-month) with normalization of erythropoiesis and iron metabolism. VCNs were proportional to chimeric hemoglobin (human α/mouse β) levels and concurrently decreased or absent HbH (Fig1B-C-D). Additionally, we tested ALS20αI in erythroid progenitors derived from CD34 cells isolated from patients with deletional and non-deletional HbH disease. Preliminary data demonstrates improvement in α-globin/β-globin mRNA ratio and reduction in formation of HbH by HPLC. In summary, we have developed new mouse models for α-thal with a lethal and reproducible phenotype. Remarkably, use of LNP technology accelerated the generation of α-thal mice and their characterization. Furthermore, our new vector ALS20αI rescued these animals with stable expression of the human α-globin gene. These data strongly support the use of ALS20αI for the cure of severe forms of α-thal, as 2 copies may be sufficient to rescue patients affected by BHFS, while 1 copy may be sufficient to cure patients affected by HbH disease. HP, SR corresponding Authors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Functional Characterization of Cooperating MGA Deletions in RUNX1::RUNX1T1 Acute Myeloid Leukemia

Max-gene associated (MGA) is a dual-specificity transcription factor that is recurrently mutated in 2-5% of all cancers and commonly seen in lung adenocarcinoma, endometrial carcinoma, and colorectal cancer. Our group has previously described heterozygous loss-of-function mutations in 9% of RUNX1::RUNX1T1 pediatric AMLs. Similar loss-of-function mutations are seen in other hematopoietic malignancies, including chronic lymphocytic leukemia (CLL), natural killer/T-cell lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), and T-cell acute lymphoblastic leukemia (T-ALL). MGA is a component of the non-canonical polycomb repressive complex (ncPRC1.6) containing an amino-terminal T-box domain and a carboxy-terminal Myc-like basic helix-loop-helix (bHLH) domain which are used to regulate MAX-network and T-box family targets. In hematological malignancies such as RUNX1::RUNX1T1 AMLs, MGA mutations disrupt or remove the Myc-like bHLH domain. The role of MGA in normal hematopoietic regulation and leukemic development are understudied. This is likely due to the lack of in vivo MGA deficiency models which are embryonic lethal. We hypothesize that the loss of MGA leads to disruption of the ncPRC1.6 complex, causing a loss in global transcriptional repression of many pathways, including Myc and E2F target genes, leading to enhanced hematopoietic cell growth and self-renewal, as well as shortening the latency of AML driven by RUNX1::RUNXT1. To test this hypothesis, we designed a multidisciplinary approach to characterize the hematopoietic function of MGA. IP-Westerns of overexpressed flag-tagged MGA and its subsequent variants in K562 cells revealed that MGA truncations disrupt interactions with components of the ncPRC1.6 complex. Because these truncations lead to the loss of MGA's molecular function, we next generated CRISPR/Cas9 to knockout MGA in Molm13 cells. Using RNA-Seq and CUT&RUN to assess the genomic localization of MGA, we determined that MGA binds to an average of 85,119 peaks and 17.7% of those sites are at transcription start sites, including cell cycle genes such as CCND2. Deletion of MGA led to the removal of repressive H3K27me3 marks and an increase in MYC binding as well as active histone marks such as H3K4me3 and H3K27Ac at the transcription start site of CCND2. This led to an increase in CCND2 expression (logFC 1.56, FDR 1.56E-12) which is supported by in vitro EdU assays showing a 10.43% increase in cells cycling into S-phase. To further define the hematopoietic function of MGA, we designed a conditional knockout Mga mouse model with LoxP sites flanking exon 3, leading to deletion of Mga in hematopoietic stem and progenitor cells (HSPCs) when crossed with Vav1-cre. Transcriptome profiling analysis of HSPCs isolated from heterozygous (Mga+/) and homozygous (Mga-/-) mice reveals that Mga-/- mice, but not Mga+/-, leads to up-regulation of cell cycle (FDR 4.2E-06), Myc (FDR 6.3E-10), and E2f (FDR 2.9E-17) signaling pathways. Ex vivo serial CFU replating and cell proliferation assays support this, showing that Mga-/- leads to increased stem cell self-renewal and cell growth. However, bone marrow and spleen analysis of both Mga+/- and Mga-/- mice reveal no changes in immunophenotype when compared to WT controls. Expression of RUNX1::RUNX1T1 isoform 9a (AML-ETO9a) in Mga+/- or Mga-/- HSPCs reveals a significant increase (69% and 74% respectively, p<0.01) in leukemic stem cell self-renewal, when compared to WT controls expressing AML-ETO9a at week 4. When transplanted in mice, Mga+/- and Mga-/- HSPCs expressing AML-ETO9a led to a rapid expansion of blasts (GFP+, lin-, cKit+) found in the peripheral blood as well as a shortened latency compared to wild-type controls expressing AML-ETO9a (Figure 1). These studies demonstrated MGA as a regulator of hematopoietic cell proliferation (Figure 2), and how MGA deficiency cooperates with oncogenes to create more aggressive leukemia. Because MGA mutations occur in many tumor types, these data also provide functional insights into ncPRC1.6 activity in leukemia and other tumor types with recurrent mutations in MGA or other components of this complex. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

ODP297 Analysis of the responsible region of TRH in the hypothalamus-pituitary-thyroid axis using the Paraventricular nucleus-specific TRH deficient mice.

Abstract Objectives Thyrotropin-releasing hormone (TRH) was the first hypothalamic hormone isolated that stimulates pituitary thyroid-stimulating hormone (TSH) secretion. TRH was later found to be distributed throughout the brain, gastrointestinal tract and pancreaticβ-cells. We previously reported the TRH null mice (conventional TRHKO) which exhibit characteristic tertiary hypothyroidism, however the responsible regionfor the hypothalamic-pituitary-thyroid (HPT) axis remained obscure because of the broad expression of TRH in hypothalamus. Previous studies suggested that TRH in the hypothalamic paraventricular nucleus (PVN) was secreted through the median eminence (ME), however it was not directly demonstrated . To determine the region functionally responsible for the HPT axis, we established and analyzed PVN-specific TRH knock-out (PVN-TRHKO) mice. Methods For the targeting vector, a Neo-loxP cassette was inserted into the 3′-UTR of the Trh gene in ES cells. Another loxP site was placed at intron 1. After we obtained the Trh +/lox [neo+] founder mice, we crossed them with the CAG-FLPe transgenic mice to remove the Neo cassette. The offspring were backcrossed to wild-type mice, producing conditional knock-out mice without the Neo cassette, which were labeled as Trh +/lox . We crossed Trh +/lox with transgenic mice expressing Single-minded homolog (Sim)1-Cre recombinase and created PVN-TRHKO mice (Sim1-Cre; Trh lox/lox mice). We confirmed that most Sim1 was expressed in the PVN using Sim1-Cre/tdTomato mice. We performed the following experiments using PVN-TRHKO andTrh lox/lox mice as control (Ct). 1) immunostaining of TRH using antibody forproTRH in cerebrum, 2)qPCR analysis detecting the expression of preproTrh mRNA in hypothalamic PVN, 3) measurement of the level of serum free-T4 and TSH, 4)qPCR analysis detecting theexpression levels of Tshβ, Prl, Cga, Gh, Pomc and Lhβ gene mRNAin the anterior pituitary and 5)immunohistochemical analysis with antibodies for TSHβ and Prolactin in anterior pituitary. Results In the PVN-TRHKO mice, 1) ProTRH-immunopositive cells were lost in the area of PVN and ME, whereas proTRH detected in the medial preoptic area(MPA) and medial preoptic nucleus (MPO) in the hypothalamus.2)The expression level of proTRHmRNA was decreased to7.7% compared with Ct.3) Free-T4 levels decreased to 50% and the TSH levels increased to 170% of those in Ct. 4) TshβmRNA levels were decreased by 65% and thePrlmRNA levels were reduced to 55% of those in Ct. 5) The number of TSH-positive andProlactin-positive cells in the pituitary significantly decreased to 50% and 60% of those in Ct. Conclusions PVN-TRHKO mice exhibited tertiary hypothyroidism similar to conventional TRHKO. The pathway of TRH neuron from PVN to ME was lost in the PVN-TRHKO mice. These findings are conclusive evidence that the TRH neuron in the PVN is the center of the HPT axis. Presentation: No date and time listed

Expression of connexin 43 protein in cardiomyocytes of heart failure mouse model.

Heart failure (HF) is the end stage of various cardiovascular diseases, with high morbidity and mortality, and is associated with a poor prognosis. One of the primary causes of HF is aortic valve disease, manifested by progressive aortic valve stenosis (AVS), resulting in increased left ventricular load, ventricular hypertrophy, ultimately ventricular dysfunction, and HF. Early assessment of the degree of cardiomyopathy and timely intervention is expected to improve patients’ cardiac function and delay or even avoid the occurrence of HF. The Wnt signaling pathway is mainly involved in regulating myocardial insufficiency after valve stenosis. Connexin 43 protein (Cx43) is an essential target of Wnt signaling pathway that forms gap junction (GJ) structures and is widely distributed in various organs and tissues, especially in the heart. The distribution and transformation of Cx43 among cardiac cells are crucial for the development of HF. To specifically label Cx43 in vivo, we established a new Cx43-BFP-GFP mouse model with two loxp sites on both sides of the tag BFP-polyA box, which can be removed by Cre recombination. This double-reporter line endowed us with a powerful genetic tool for determining the area, spatial distribution, and functional status of Cx43. It also indicated changes in electrical conduction between cells in a steady or diseased state.