Mouse Genome Research Articles

Efficient single copy integration via homology-directed repair (scHDR) by 5'modification of large DNA donor fragments in mice.

CRISPR/Cas-based approaches have largely replaced conventional gene targeting strategies. However, homology-directed repair (HDR) in the mouse genome is not very efficient, and precisely inserting longer sequences using HDR remains challenging given that donor constructs preferentially integrate as concatemers. Here, we showed that injecting 5' biotinylated donor DNA into mouse embryos at the two-cell stage led to efficient single-copy HDR (scHDR) allele generation. Our dedicated genotyping strategy showed that these alleles occurred with frequencies of 19%, 20%, and 26% at three independent gene loci, indicating that scHDR was dramatically increased by 5' biotinylation. Thus, we suggest that the combination of a 5' biotinylated donor and diligent analysis of concatemer integration are prerequisites for efficiently and reliably generating conditional alleles or other large fragment knock-ins in the mouse genome.

Mitochondrial carbonic anhydrase VA and VB: properties and roles in health and disease.

Carbonic anhydrase V (CAV), a mitochondrial enzyme, was first isolated from guinea-pig liver and subsequently identified in mice and humans. Later, studies revealed that the mouse genome contains two mitochondrial CA sequences, named Car5A and Car5B. The CAVA enzyme is most highly expressed in the liver, whereas CAVB shows a broad tissue distribution. Car5A knockout mice demonstrated a predominant role for CAVA in ammonia detoxification, whereas the roles of CAVB in ureagenesis and gluconeogenesis were evident only in the absence of CAVA. Previous studies have suggested that CAVA is mainly involved in the provision of HCO3 - for biosynthetic processes. In children, mutations in the CA5A gene led to reduced CA activity, and the enzyme was sensitive to increased temperature. The metabolic profiles of these children showed a reduced supply of HCO3 - to the enzymes that take part in intermediary metabolism: carbamoylphosphate synthetase, pyruvate carboxylase, propionyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase. Although the role of CAVB is still poorly understood, a recent study reported that it plays an essential role in human Sertoli cells, which sustain spermatogenesis. Metabolic disease associated with CAVA appears to be more common than other inborn errors of metabolism and responds well to treatment with N-carbamyl-l-glutamate. Therefore, early identification of hyperammonaemia will allow specific treatment with N-carbamyl-l-glutamate and prevent neurological sequelae. Carbonic anhydrase VA deficiency should therefore be considered a treatable condition in the differential diagnosis of hyperammonaemia in neonates and young children.

Reliable and Fast Genotyping Protocol for Galactosylceramidase (Galc) in the Twitcher (Twi) Mouse.

Twitcher (Twi) is a neurological Krabbe disease (KD, or globoid cell leukodystrophy) spontaneous mutant line in mice. The genome of the Twi mouse presents a single nucleotide polymorphism (SNP), leading to an enzymatically inactive galactosylceramidase (Galc) protein that causes KD. In this context, mouse Twi genotyping is an essential step in KD research. To date, the genotyping method used is labor-intensive and often has ambiguous results. Here, we evaluated a novel protocol for the genotype determination of Galc mutation status in Twi mice based on the allele-discrimination real-time polymerase chain reaction (PCR). Here, DNA is extracted from Twi mice (n = 20, pilot study; n = 120, verification study) and control group (n = 10, pilot study; n = 30 verification study) and assessed by allele-discrimination real-time PCR to detect SNP c.355G>A. Using the allele-discrimination PCR, all of the samples are identified correctly with the genotype GG (wild-type, WT), GA (heterozygote, HET), or AA (homozygote, HOM) using the first analysis and no animals are not genotyped. We demonstrated that this novel method can be used to distinguish KD timely, accurately, and without ambiguity in HOM, WT, and HET animals. This protocol represents a great opportunity to increase accuracy and speed in KD research.

The Clu‐rs2279590_h2kb variant increases axonal and neuritic damage in 5xFAD mice

AbstractBackgroundGenome‐Wide Association Studies (GWAS) implicate clusterin (CLU, also known as apolipoprotein J) as a risk factor for late‐onset Alzheimer's disease (LOAD), with elevated expression of CLU being associated with increased risk of LOAD. The rs2279590 SNP risk allele is located within a putative enhancer element in intron 7 of CLU. Indeed, deletion of a 115bp region including this SNP reduces expression of CLU and neighboring genes (EPHX2 and PTK2B) on Chromosome 8. The region surrounding rs2279590 is poorly conserved within the mouse genome. Consequently, to model the effect of the rs2279590 risk allele on development of AD‐related pathology in mice we developed mice with a partially humanized allele of Clu.MethodCRISPR was used to substitute a 2kb region of human CLU (intron 7 to exon 9) containing the enhancer region and the GWAS AD‐risk rs2279590 SNP variant for the orthologous region of Clu within the C57BL/6J mouse genome. Clu‐rs2279590_h2kb mice were crossed with 5xFAD mice and aged to 4 months to study the effects of the partially humanized Clu allele on development of AD‐related pathologies in 5xFAD mice. Coronal brain sections were immunolabeled to visualize dense‐core plaques (Thioflavin‐S), microglia (IBA1), reactive astrocytes (GFAP), axonal and neuritic damage (neurofilament light chain (NfL) and LAMP1, respectively). Confocal images of hippocampal and cortical regions were analyzed using Imaris software. In addition, Aβ and NfL levels were quantified in the brain tissue and plasma NfL measured using meso scale discovery (MSD) technology.ResultFour‐month‐old 5xFAD hemizygous, Clu‐rs2279590_h2kb homozygous mice had reduced microglial density and volume compared to 5xFAD hemizygous controls. No change was found in astrocyte density or reactivity. Despite comparable plaque load between 5xFAD and 5xFAD x Clu‐rs2279590_h2kb mice, neuritic and axonal damage was elevated in the brain of 5xFAD x Clu‐rs2279590_h2kb mice compared to 5xFAD, while plasma NfL level did not differ. Insoluble Aβ42 levels mirrored plaque density.ConclusionDespite the lack of impact on Aβ plaque deposition, and moderate influence on gliosis, four‐month‐old 5xFAD hemizygous, Clu‐rs2279590_h2kb homozygous mice display elevated axonal and neuritic damage compared to 5xFAD hemizygous controls.

An AGS-associated mutation in ADAR1 catalytic domain results in early-onset and MDA5-dependent encephalopathy with IFN pathway activation in the brain

BackgroundAicardi–Goutières syndrome (AGS) is a severe neurodegenerative disease with clinical features of early-onset encephalopathy and progressive loss of intellectual abilities and motor control. Gene mutations in seven protein-coding genes have been found to be associated with AGS. However, the causative role of these mutations in the early-onset neuropathogenesis has not been demonstrated in animal models, and the mechanism of neurodegeneration of AGS remains ambiguous.MethodsVia CRISPR/Cas-9 technology, we established a mutant mouse model in which a genetic mutation found in AGS patients at the ADAR1 coding gene (Adar) loci was introduced into the mouse genome. A mouse model carrying double gene mutations encoding ADAR1 and MDA-5 was prepared using a breeding strategy. Phenotype, gene expression, RNA sequencing, innate immune pathway activation, and pathologic studies including RNA in situ hybridization (ISH) and immunohistochemistry were used for characterization of the mouse models to determine potential disease mechanisms.ResultsWe established a mouse model bearing a mutation in the catalytic domain of ADAR1, the D1113H mutation found in AGS patients. With this mouse model, we demonstrated a causative role of this mutation for the early-onset brain injuries in AGS and determined the signaling pathway underlying the neuropathogenesis. First, this mutation altered the RNA editing profile in neural transcripts and led to robust IFN-stimulated gene (ISG) expression in the brain. By ISH, the brains of mutant mice showed an unusual, multifocal increased expression of ISGs that was cell-type dependent. Early-onset astrocytosis and microgliosis and later stage calcification in the deep white matter areas were observed in the mutant mice. Brain ISG activation and neuroglial reaction were completely prevented in the Adar D1113H mutant mice by blocking RNA sensing through deletion of the cytosolic RNA receptor MDA-5.ConclusionsThe Adar D1113H mutation in the ADAR1 catalytic domain results in early-onset and MDA5-dependent encephalopathy with IFN pathway activation in the mouse brain.

ABCA7*V1599M variant in 5xFAD mice mediates differences in amyloid‐beta pathology and reactive gliosis

AbstractBackgroundIn several large genome‐wide association studies (GWAS), genetic polymorphisms of ATP‐binding cassette transporter subfamily A member 7 (Abca7) have been identified as a risk factor for late‐onset Alzheimer’s Disease (LOAD). ABCA7 is a member of a family of ATP‐binding cassette (ABC) transporters and functions as a transmembrane protein mediating the release of cholesterols and phospholipids from cellular membranes. Previous studies have also proposed its role in mediating phagocytic clearance of amyloid beta (Aβ) oligomers within macrophages in the brain.MethodTo study the effects of ABCA7 on the development of AD relevant pathologies we introduced an equivalent coding sequence change that has been identified as a risk variant for LOAD (V1613M) into the C57BL/6 mouse genome using CRISPR/Cas9 (V1599M). We set out to characterize the ABCA7V1599M variant and investigate its impact on AD pathology when crossed with 5xFAD transgenic mice, generating four distinct groups: WT, ABCA7V1599M homozygous, 5xFAD, and 5xFAD x ABCA7V1599M homozygous. Characterization was performed using histological staining and biochemical approaches.ResultCoronal brain sections from 4‐month‐old mice (n=5‐6 mice/sex/genotype) revealed significantly reduced burden of dense‐core Aβ plaques in confocal images of both subiculum and visual cortical regions of 5xFAD x ABCA7V1599M mice compared to 5xFAD mice characterized by reduced Thioflavin‐S staining. Biochemical analysis mirrored these findings and demonstrated reductions of both soluble and insoluble Aβ40 and Aβ42 levels within these same brain regions. To examine reactive gliosis to plaque burden, we immuno‐stained for ionized calcium binding adaptor molecule 1 (IBA1) and glial fibrillary protein (GFAP) to visualize microglia and reactive astrocytes, respectively. Confocal analysis revealed reduced densities of both IBA1+ microglia and GFAP+ reactive astrocytes within subiculum and cortical regions of 5xFAD x ABCA7V1599M mice compared to 5xFAD mice. We also found reduced plaque‐associated neuritic damage in 5xFAD x ABCA7V1599M mice upon immuno‐staining for lysosome‐associated membrane glycoprotein 1 (LAMP1) to examine neuritic dystrophyConclusionTogether, these results characterize the effects of the Abca7em1Aduci allele and its ABCA7V1599M missense mutation on 5xFAD‐mediated pathology, specifically in Aβ plaque development, glial morphology, and neuritic damage.

Comparison of degu (Octodon degus), Naked Mole Rat, Mouse and Human genomes identified common hallmarks of aging and Alzheimer's Disease

AbstractBackgroundTo provide new insight into the mechanisms of Alzheimer’s disease (AD) pathogenesis and aid the discovery of new therapies in humans it is important to expand and complement research using mouse models by developing, characterizing, and validating suitable new or unconventional mammalian, non‐murine models of AD. Our goal was to create genome assemblies at the chromosome level associated with AD‐like pathogenesis using two long‐lived animal models. The degu (Octodon degus) which naturally exhibits AD neuropathology and the naked mole rat (H. glaber) best known for its resistance to cancer and aging. To understand evolutionary paths to longevity and associated diseases like AD we characterized their genome features and identified common substitutions in long‐lived rodents that support enhanced tolerance to DNA damage.MethodSynteny maps among M. musculus, H. glaber, H. sapiens and O. degus were created using SyMAP 5.0.6. To evaluate disease‐related mutations in humans a pairwise comparison of protein sequences was performed using the HGMD.ResultWe identified several synteny blocks that were part of the putative rodent ancestor genome. 78% genome was covered in H. glaber and 75% coverage in O. degus. In addition, 58 of the 426 synteny blocks were longer than 10 Mbp in M. musculus and 62 in O. degus. The identification of substitutions in orthologs at disease‐causing sites revealed that DNA damage and repair genes are under selection in long‐lived species. Our comparative genomic analyses identified that the top disease categories enriched in degu and naked mole rat were associated with lipid metabolism, DNA repair, stress and IGF‐1.ConclusionOur comparative genome analysis revealed changes in genes and pathways associated with adaptations to acquired longevity. Substitutions at AD causing sites may contribute to long‐term survival pathways affected by natural selection in long‐lived models. When compared to short‐lived mouse models, pathways associated with lipid metabolism, oxidative stress and DNA repair were identified. This data is particularly valuable for providing a within family (Rodentia) comparison for mouse AD model genomics to develop a comprehensive analysis to determine conserved genetic alterations between the unconventional animal models and to bridge our existing databases of mouse models and humans.

Roles and Implications of Palindromic DNA in Alzheimer's Disease: A Systematic Review and Computational Analysis

AbstractBackgroundA palindrome in DNA is a sequence consisting of two adjacent identical or highly similar inverted repeats. Palindromes are distributed throughout the genome and play important roles in gene expression and regulation. Palindromic mutations are linked to many disease states, such as neuronal disorders, mental retardation, and various cancers.MethodA systematic review was conducted in which existing literature on the palindromic DNA within disease‐associated loci of AD is synthesised. Subsequent computational analysis of DNA palindromes within promoters, introns, and exons were characterised and discussed.ResultStudies included in the review identified palindromes and palindrome‐altering in the causative genes associated with AD, which may influence the fine tuning of APP and PSEN gene expression and/or alternative splicing of the transcript, leading to their different susceptibilities to beta‐amyloidosis. Results of computational analysis suggested roles in transcription factor binding and regulatory control within promoters, alongside implications for vulnerability of palindromic regions to genomic variation within introns. Mutations in intronic regions may introduce novel regulatory activity and affect splice sites. Comparisons with the mice genome showed differential tolerance of longer unstable palindromic sequences.ConclusionOverall, the results from this study serve as a resource of identified AD‐associated palindromic sequences, which could affect various cellular processes leading to gene dysregulation and AD pathogenesis. Further research is warranted to enlighten their roles in the specific disease mechanism.

GENCODE: reference annotation for the human and mouse genomes in 2023.

GENCODE produces high quality gene and transcript annotation for the human and mouse genomes. All GENCODE annotation is supported by experimental data and serves as a reference for genome biology and clinical genomics. The GENCODE consortium generates targeted experimental data, develops bioinformatic tools and carries out analyses that, along with externally produced data and methods, support the identification and annotation of transcript structures and the determination of their function. Here, we present an update on the annotation of human and mouse genes, including developments in the tools, data, analyses and major collaborations which underpin this progress. For example, we report the creation of a set of non-canonical ORFs identified in GENCODE transcripts, the LRGASP collaboration to assess the use of long transcriptomic data to build transcript models, the progress in collaborations with RefSeq and UniProt to increase convergence in the annotation of human and mouse protein-coding genes, the propagation of GENCODE across the human pan-genome and the development of new tools to support annotation of regulatory features by GENCODE. Our annotation is accessible via Ensembl, the UCSC Genome Browser and https://www.gencodegenes.org.

TDP-43 safeguards the embryo genome from L1 retrotransposition.

Transposable elements (TEs) are genomic parasites that propagate within the host genome and introduce mutations. Long interspersed nuclear element-1 (LINE-1 or L1) is the major TE class, which occupies nearly 20% of the mouse genome. L1 is highly active in mammalian preimplantation embryos, posing a major threat to genome integrity, but the mechanism of stage-specific protection against L1 retrotransposition is unknown. Here, we show that TAR DNA-binding protein 43 (TDP-43), mutations in which constitute a major risk factor for amyotrophic lateral sclerosis, inhibits L1 retrotransposition in mouse embryonic stem cells (mESCs) and preimplantation embryos. Knockdown of TDP-43 resulted in massive genomic L1 expansion and impaired cell growth in preimplantation embryos and ESCs. Functional analysis demonstrated that TDP-43 interacts with L1 open reading frame 1 protein (L1 ORF1p) to mediate genomic protection, and loss of this interaction led to derepression of L1 retrotransposition. Our results identify TDP-43 as a guardian of the embryonic genome.

295 Genotype-phenotype correlation analysis in recessive dystrophic epidermolysis bullosa using in situ germline mouse genome editing technique

Recessive dystrophic epidermolysis bullosa (RDEB) is an intractable skin genetic disease characterized by the skin fragility leading to the recurrent blistering. The mutations in COL7A1 gene, encoding type VII collagen (C7) that forms anchoring fibrils, cause RDEB. RDEB patients are known to show variable prognosis, likely influenced by the position of pathogenic mutations. However, as the mutations in COL7A1 gene distribute across the gene without any apparent hotspots and the majority of RDEB patients harbor the mutations in a compound heterozygous manner, the genotype-phenotype correlation is not fully elucidated.

Abstract 11608: Differences in Genetic Variants and Common Genetic Regulators between Arrhythmogenic Cardiomyopathy and Arrhythmogenic Dilated Cardiomyopathy Identified Using Systems Genetics Approaches

Introduction: Prominent arrhythmias are features of arrhythmogenic cardiomyopathy (ACM) and arrhythmogenic dilated cardiomyopathy (aDCM). Despite clinicopathological differences, these diseases share common genetic causes. Hypothesis: This study aimed to identify variants in ACM and aDCM and map the candidate genetic regulators using a mouse genetic reference population (GRP). Methods: Whole exome sequencing was performed on 226 individuals with ACM and 102 patients with aDCM. Bioinformatics analysis was applied to identify the pathogenic or likely pathogenic variants followed by direct sequencing confirmation. Expression quantitative trait loci (eQTLs) mapping to heart transcriptomes of GRP of BXD recombinant inbred (RI) mice was conducted in GeneNetwork (http://www.genenetwork.org). Results: In the ACM cohort, a total of 82 variants (MAF<0.01) in 24 genes have been confirmed being positive in 42 probands. Depending on the frequency of the identified variants, the top 10 genes (excluding TTN) with the most pathogenic variants were PKP2, MYH6, FLNC, RYR2, MYH7B, PLEC, JPH2, SYNE1, NRAP and POSTN. In the aDCM cohort, 141 pathogenic variants have been identified and 91 patients harbored at least 1 deleterious variant. The top 10 genes were RYR2, FLNC, MYH7, DSP, SCN5A,MYPN, NEBL, CTNNA3, ACAP6 and SLC22A5. We analyzed the distribution of the first principal component (PC1) of mRNA expression levels of those 10 genes (eigentraits) from each cohort in the heart across BXD strains. Interval mapping for PC1 eigentraits for ACM identified a significant eQTL (P<0.05) in the mouse genome at 24-36 Mb of chromosome (Chr) 6. In aDCM, interval mapping for PC1 eigentraits identified 3 suggestive eQTLs at Chr 5 (44-53 Mb), Chr 6 (32-34 Mb) and Chr 15 (82-84 Mb). Further gene exploration of these eQTLs suggested that Mtpn, Lep, Cyb5r3, Tspo, Slit2, and Ppargc1a could be common regulators that interact with those top ACM and aDCM genes. Conclusions: This study identified diverse genes in ACM and aDCM cases. Our joint human and mouse analysis using a systems genetics approach identified genetic regulators that could affected expression of genes with pathogenic variants in ACM and aDCM patients.

Dynamic DNA methylation reveals novel cis-regulatory elements in mouse hematopoiesis

Differentiation of hematopoietic stem and progenitor cells to terminally differentiated immune cells is accompanied by large-scale remodeling of the DNA methylation landscape. Although significant insights into the molecular mechanisms of hematopoietic tissue regeneration were derived from mouse models, profiling of DNA methylation has been hampered by high cost or low resolution using available methods. The recent development of the Infinium Mouse Methylation BeadChip (MMBC) array facilitates methylation profiling of the mouse genome at a single CpG resolution at affordable cost. We extended the RnBeads package to provide a computational framework for the analysis of MMBC data. This framework was applied to a newly generated reference map of mouse hematopoiesis encompassing nine different cell types. Analysis of dynamically regulated CpG sites showed progressive and unidirectional DNA methylation changes from hematopoietic stem and progenitor cells to differentiated hematopoietic cells and allowed the identification of lineage- and cell type-specific DNA methylation programs. Comparison with previously published catalogs of cis-regulatory elements (CREs) revealed 12,856 novel putative CREs that were dynamically regulated by DNA methylation (mdCREs). These mdCREs were predominantly associated with patterns of cell type-specific DNA hypomethylation and could be identified as epigenetic control regions regulating the expression of key hematopoietic genes during differentiation. In summary, we established an analysis pipeline for MMBC data sets and provide a DNA methylation atlas of mouse hematopoiesis. This resource allowed us to identify novel putative CREs involved in hematopoiesis and will serve as a platform to study epigenetic regulation of normal and malignant hematopoiesis.

Long-term monitoring of intravital biological processes using fluorescent protein-assisted NIR-II imaging

High spatial resolution, low background, and deep tissue penetration have made near-infrared II (NIR-II) fluorescence imaging one of the most critical tools for in vivo observation and measurement. However, the relatively short retention time and potential toxicity of synthetic NIR-II fluorophores limit their long-term application. Here, we report the use of infrared fluorescent proteins (iRFPs) as in vitro and in vivo NIR-II probes permitting prolonged continuous imaging (up to 15 months). As a representative example, iRFP713 is knocked into the mouse genome to generate a transgenic model to allow temporal and/or spatial expression control of the probe. To demonstrate its feasibility in a genuine diagnostic context, we adopt two liver regeneration models and successfully track the process for a week. The performance and monitoring efficacy are comparable to those of μCT and superior to those of indocyanine green dye. We are also able to effectively observe the pancreas, despite its deep location, under both physiological and pathological conditions. These results indicate that the iRFP-assisted NIR-II fluorescence system is suitable for monitoring various tissues and in vivo biological processes, providing a powerful noninvasive long-term imaging platform.

DNA sequence and chromatin modifiers cooperate to confer epigenetic bistability at imprinting control regions

Genomic imprinting is regulated by parental-specific DNA methylation of imprinting control regions (ICRs). Despite an identical DNA sequence, ICRs can exist in two distinct epigenetic states that are memorized throughout unlimited cell divisions and reset during germline formation. Here, we systematically study the genetic and epigenetic determinants of this epigenetic bistability. By iterative integration of ICRs and related DNA sequences to an ectopic location in the mouse genome, we first identify the DNA sequence features required for maintenance of epigenetic states in embryonic stem cells. The autonomous regulatory properties of ICRs further enabled us to create DNA-methylation-sensitive reporters and to screen for key components involved in regulating their epigenetic memory. Besides DNMT1, UHRF1 and ZFP57, we identify factors that prevent switching from methylated to unmethylated states and show that two of these candidates, ATF7IP and ZMYM2, are important for the stability of DNA and H3K9 methylation at ICRs in embryonic stem cells.

RF34 | PMON203 Copy Number Variant Analysis Reveals Novel Candidate Genes Associated with Primary Ovarian Insufficiency

Abstract Primary ovarian insufficiency (POI) is characterized by a heterogeneous genetic background. Moreover, the etiology of most POI cases remains unclear. Herein, our goal was to investigate the presence of rare deletions or duplications in patients presenting with POI, which could lead to identification of novel chromosomal regions and/or genes potentially associated with disease risk. Patients and Methods Sixty-six patients presenting idiopathic POI were evaluated; 44 had primary amenorrhea and 22 secondary amenorrhea. Chromosomal microarray analysis was performed to detect copy number variants (CNVs) using array-CGH or SNP-array. Identified CNVs were validated by population databases of either health individuals (Database of Genomic Variants - DGV) or patients carrying chromosomal structural alterations (DECIPHER). A set of rare CNVs was selected and annotated in regard to gene content and clinical associations using bioinformatic tools (RefSeq, OMIM, PubMed, Ovarian Kaleidoscope Database, and Mouse Genome Informatics). Results Fifteen rare CNVs were found in 13 patients, ten of them with primary amenorrhea. The identified CNVs comprised four deleted and 11 duplicated regions, in which the break points occurred intragenic in 8 of them. Most of the CNVs were mapped in the autosomes. Seven CNVs identified in 5 patients were classified as pathogenic or probably pathogenic; three were novel variants containing candidate genes associated with ovarian development [19q13.43 (NLRP5) and Xq13.2 (XIST)] or function [(2q37.3 (PER2)]. The other pathogenic CNVs were previously implicated in POI etiology; two of them [Xq27.2q28 del (14.5 Mb) and Xq22.1q27.1 dup (41.4 Mb)] were encompassed in a complex chromosome rearrangement, associated with 20p13 dup (2.7 Mb), and the last region was 15q25.2 deletion. Four VUS containing genes associated with ovarian development [2p23.1 (XDH and EHD3), 9p24.1 (KDM4C), 22q12.1 (ZNRF3)] or function [1p31.1 (PTGFR)] were identified. Conclusion CNVs analysis indicated a genetic etiology in 10% of these POI patients. New candidate genes associated with ovarian development and function were revealed. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 12:54 p.m. - 12:59 p.m.

In Vivo Models for Prostate Cancer Research.

In 2022, prostate cancer (PCa) is estimated to be the most commonly diagnosed cancer in men in the United States-almost 270,000 American men are estimated to be diagnosed with PCa in 2022. This review compares and contrasts in vivo models of PCa with regards to the altered genes, signaling pathways, and stages of tumor progression associated with each model. The main type of model included in this review are genetically engineered mouse models, which include conditional and constitutive knockout model. 2D cell lines, 3D organoids and spheroids, xenografts and allografts, and patient derived models are also included. The major applications, advantages and disadvantages, and ease of use and cost are unique to each type of model, but they all make it easier to translate the tumor progression that is seen in the mouse prostate to the human prostate. Although both human and mouse prostates are androgen-dependent, the fact that the native, genetically unaltered prostate in mice cannot give rise to carcinoma is an especially critical component of PCa models. Thanks to the similarities between the mouse and human genome, our knowledge of PCa has been expanded, and will continue to do so, through models of PCa.

Detection of Transgene Location in the CYP2A13/2B6/2F1-transgenic Mouse Model using Optical Genome Mapping Technology.

Most transgenic mouse models are generated through random integration of the transgene. The location of the transgene provides valuable information for assessing potential effects of the transgenesis on the host and for designing genotyping protocols that can amplify across the integration site, but it is challenging to identify. Here, we report the successful utility of optical genome mapping technology to identify the transgene insertion site in a CYP2A13/2B6/2F1-transgenic mouse model, which produces three human cytochrome P450 (P450) enzymes (CYP2A13, CYP2B6, and CYP2F1) that are encoded by neighboring genes on human chromosome 19. These enzymes metabolize many drugs, respiratory toxicants, and chemical carcinogens. Initial efforts to identify candidate insertion sites by whole genome sequencing was unsuccessful, apparently because the transgene is located in a region of the mouse genome that contains highly repetitive sequences. Subsequent utility of the optical genome mapping approach, which compares genome-wide marker distribution between the transgenic mouse genome and a reference mouse (GRCm38) or human (GRCh38) genome, localized the insertion site to mouse chromosome 14, between two marker positions at 4451324 base pair and 4485032 base pair. A transgene-mouse genome junction sequence was further identified through long-polymerase chain reaction amplification and DNA sequencing at GRCm38 Chr.14:4484726. The transgene insertion (∼2.4 megabase pair) contained 5-7 copies of the human transgenes, which replaced a 26.9-33.4 kilobase pair mouse genomic region, including exons 1-4 of Gm3182, a predicted and highly redundant gene. Finally, the sequencing results enabled the design of a new genotyping protocol that can distinguish between hemizygous and homozygous CYP2A13/2B6/2F1-transgenic mice. SIGNIFICANCE STATEMENT: This study characterizes the genomic structure of, and provides a new genotyping method for, a transgenic mouse model that expresses three human P450 enzymes, CYP2A13, CYP2B6, and CYP2F1, that are important in xenobiotic metabolism and toxicity. The demonstrated success in applying the optical genome mapping technology for identification of transgene insertion sites should encourage others to do the same for other transgenic models generated through random integration, including most of the currently available human P450 transgenic mouse models.

A map of cis-regulatory modules and constituent transcription factor binding sites in 80% of the mouse genome

BackgroundMouse is probably the most important model organism to study mammal biology and human diseases. A better understanding of the mouse genome will help understand the human genome, biology and diseases. However, despite the recent progress, the characterization of the regulatory sequences in the mouse genome is still far from complete, limiting its use to understand the regulatory sequences in the human genome.ResultsHere, by integrating binding peaks in ~ 9,000 transcription factor (TF) ChIP-seq datasets that cover 79.9% of the mouse mappable genome using an efficient pipeline, we were able to partition these binding peak-covered genome regions into a cis-regulatory module (CRM) candidate (CRMC) set and a non-CRMC set. The CRMCs contain 912,197 putative CRMs and 38,554,729 TF binding sites (TFBSs) islands, covering 55.5% and 24.4% of the mappable genome, respectively. The CRMCs tend to be under strong evolutionary constraints, indicating that they are likely cis-regulatory; while the non-CRMCs are largely selectively neutral, indicating that they are unlikely cis-regulatory. Based on evolutionary profiles of the genome positions, we further estimated that 63.8% and 27.4% of the mouse genome might code for CRMs and TFBSs, respectively.ConclusionsValidation using experimental data suggests that at least most of the CRMCs are authentic. Thus, this unprecedentedly comprehensive map of CRMs and TFBSs can be a good resource to guide experimental studies of regulatory genomes in mice and humans.

Baat Gene Knockout Alters Post-Natal Development, the Gut Microbiome, and Reveals Unusual Bile Acids in Mice

Bile acids (BAs) are steroid detergents in bile that contribute to fat absorption, cell signaling, and microbiome interactions. The final step in their synthesis is amino acid conjugation with either glycine or taurine in the liver by the enzyme bile acid-CoA:amino acid N-acyltransferase (BAAT). Here, we describe the microbial, chemical, and physiological consequences of Baat gene knockout. Baat-/- mice were underweight after weaning but quickly exhibited catch-up growth. At three weeks of age, KO animals had increased phospholipid excretion and decreased subcutaneous fat pad mass, liver mass, glycogen staining in hepatocytes, and hepatic vitamin A stores, but these were less marked in adulthood. Additionally, KO mice had an altered microbiome in early life. Their BA pool was highly enriched in cholic acid but not completely devoid of conjugated BAs. KO animals had 27-fold lower taurine-conjugated BAs than wild type in their liver but similar concentrations of glycine-conjugated BAs and higher microbially conjugated BAs. Furthermore, the BA pool in Baat-/- was enriched in a variety of unusual BAs that were putatively sourced from cysteamine conjugation with subsequent oxidation and methylation of the sulfur group mimicking taurine. Antibiotic treatment of KO mice indicated the microbiome was not the likely source of the unusual conjugations, instead, the unique BAs in KO animals were likely derived from the peroxisomal acyltransferases Acnat1 and Acnat2, which are duplications of Baat in the mouse genome that are inactivated in humans. This study demonstrates that BA conjugation is important for early life development of mice.