Generation of a DAT-P2A-Flpo mouse line for intersectional genetic targeting of dopamine neuron subpopulations.

Dopaminergic projections exert widespread influence over multiple brain regions and modulate various behaviors including movement, reward learning, and motivation. It is increasingly appreciated that dopamine neurons are heterogeneous in their gene expression, circuitry, physiology, and function. Current approaches to target dopamine neurons are largely based on single gene drivers, which either label all dopamine neurons or mark a subset but concurrently label non-dopaminergic neurons. Here, we establish a mouse line with Flpo recombinase expressed from the endogenous Slc6a3 (dopamine active transporter [DAT]) locus. DAT-P2A-Flpo mice can be used together with Cre-expressing mouse lines to efficiently and selectively label dopaminergic subpopulations using Cre/Flp-dependent intersectional strategies. We demonstrate the utility of this approach by generating DAT-P2A-Flpo;NEX-Cre mice that specifically label Neurod6-expressing dopamine neurons, which project to the nucleus accumbens medial shell. DAT-P2A-Flpo mice add to a growing toolbox of genetic resources that will help parse the diverse functions mediated by dopaminergic circuits.

Male-biased fasting-induced changes in hepatic tauro-cholic acid and plasma cholesterol in Sult2a8-haplodeficient mice.

SULT2A8 is a male-predominant and liver-specific mouse cytosolic sulfotransferase (SULT) that sulfonates 7α-hydroxyl (7α-OH) bile acids in vitro. Sulfonation regulates bile acid homeostasis, which in turn regulates cholesterol and energy metabolism. Using the Sult2a8-heterozygous (HT) mouse model created earlier in our laboratory, we aimed to investigate the physiological role of SULT2A8 in sulfonating 7α-OH bile acids and its impact on energy metabolism in vivo under both fed and energy-deprivation conditions. Disruption of one allele of the Sult2a8 gene in male HT mice resulted in losing ~ 50% of the 7α-OH sulfonating activity compared to wild-type (WT) control, but no significant change in female HT mice. Under the fed condition comparing the levels of hepatic and biliary bile acids as well as plasma/serum energy metabolites, HT mice displayed a profile similar to that of WT mice, suggesting that the Sult2a8-haplodeficient mice conducted normal energy metabolism. However, after 48-h fasting, a significant decrease in plasma cholesterol level was found in male HT mice but without any significant reduction in female HT mice. Of interest, in male Sult2a8-haplodeficient mice, an increase of the hepatic taurine-conjugated cholic acid level was noted but no noticeable change in other tested bile acids after fasting. Taken together, SULT2A8 is a male-specific and key hepatic SULT in metabolizing 7α-OH primary bile acids. During energy deprivation, SULT2A8 is required to maintain the bile acid and cholesterol metabolism, suggesting SULT is a potential therapeutic target for controlling metabolic diseases.

Aifm2, a NADH Oxidase, Supports Robust Glycolysis and Is Required for Cold- and Diet-Induced Thermogenesis.

Brown adipose tissue (BAT) is highly metabolically active tissue that dissipates energy via UCP1 as heat, and BAT mass is correlated negatively with obesity. The presence of BAT/BAT-like tissue in humans renders BAT as an attractive target against obesity and insulin resistance. Here, we identify Aifm2, a NADH oxidoreductase domain containing flavoprotein, as a lipid droplet (LD)-associated protein highly enriched in BAT. Aifm2 is induced by cold as well as by diet. Upon cold or ß-adrenergic stimulation, Aifm2 associates with the outer side of the mitochondrial inner membrane. As a unique BAT-specific first mammalian NDE (external NADH dehydrogenase)-like enzyme, Aifm2 oxidizes NADH to maintain high cytosolic NAD levels in supporting robust glycolysis and to transfer electrons to the electron transport chain (ETC) for fueling thermogenesis. Aifm2 in BAT and subcutaneous white adipose tissue (WAT) promotes oxygen consumption, uncoupled respiration, and heat production during cold- and diet-induced thermogenesis. Aifm2, thus, can ameliorate diet-induced obesity and insulin resistance.

IRG1 and Inducible Nitric Oxide Synthase Act Redundantly with Other Interferon-Gamma-Induced Factors To Restrict Intracellular Replication of Legionella pneumophila.

Interferon gamma (IFN-γ) restricts the intracellular replication of many pathogens, but the mechanism by which IFN-γ confers cell-intrinsic pathogen resistance remains unclear. For example, intracellular replication of the bacterial pathogen Legionella pneumophila in macrophages is potently curtailed by IFN-γ. However, consistent with prior studies, no individual genetic deficiency that we tested completely abolished IFN-γ-mediated control. Intriguingly, we observed that the glycolysis inhibitor 2-deoxyglucose (2DG) partially rescued L. pneumophila replication in IFN-γ-treated macrophages. 2DG inhibits glycolysis and triggers the unfolded protein response, but unexpectedly, it appears these effects are not responsible for perturbing the antimicrobial activity of IFN-γ. Instead, we found that 2DG rescues bacterial replication by inhibiting the expression of two key antimicrobial factors, inducible nitric oxide synthase (iNOS) and immune-responsive gene 1 (IRG1). Using immortalized and primary macrophages deficient in iNOS and IRG1, we confirmed that loss of both iNOS and IRG1, but not individual deficiency in either gene, partially reduced IFN-γ-mediated restriction of L. pneumophila Further, using a combinatorial CRISPR/Cas9 mutagenesis approach, we found that mutation of iNOS and IRG1 in combination with four other genes (CASP11, IRGM1, IRGM3, and NOX2) resulted in a total loss of L. pneumophila restriction by IFN-γ in primary bone marrow macrophages. Our study defines a complete set of cell-intrinsic factors required for IFN-γ-mediated restriction of an intracellular bacterial pathogen and highlights the combinatorial strategy used by hosts to block bacterial replication in macrophages.IMPORTANCELegionella pneumophila is one example among many species of pathogenic bacteria that replicate within mammalian macrophages during infection. The immune signaling factor interferon gamma (IFN-γ) blocks L. pneumophila replication in macrophages and is an essential component of the immune response to L. pneumophila and other intracellular pathogens. However, to date, no study has identified the exact molecular factors induced by IFN-γ that are required for its activity. We generated macrophages lacking different combinations of IFN-γ-induced genes in an attempt to find a genetic background in which there is a complete loss of IFN-γ-mediated restriction of L. pneumophila We identified six genes that comprise the totality of the IFN-γ-dependent restriction of L. pneumophila replication in macrophages. Our results clarify the molecular basis underlying the potent effects of IFN-γ and highlight how redundancy downstream of IFN-γ is key to prevent exploitation of macrophages by pathogens.

CRISPR-READI: Efficient Generation of Knockin Mice by CRISPR RNP Electroporation and AAV Donor Infection.

Genetically engineered mouse models harboring large sequence insertions or modifications are critical for a wide range of applications including endogenous gene tagging, conditional knockout, site-specific transgene insertion, and gene replacement; however, existing methods to generate such animals remain laborious and costly. To address this, we developed an approach called CRISPR-READI (CRISPR RNP electroporation and AAV donor infection), combining adeno-associated virus (AAV)-mediated HDR donor delivery with Cas9/sgRNA RNP electroporation to engineer large site-specific modifications in the mouse genome with high efficiency and throughput. We successfully targeted a 774 bp fluorescent reporter, a 2.1 kb CreERT2 driver, and a 3.3 kb expression cassette into endogenous loci in both embryos and live mice. CRISPR-READI is applicable to most widely used knockin schemes requiring donor lengths within the 4.9 kb AAV packaging capacity. Altogether, CRISPR-READI is an efficient, high-throughput, microinjection-free approach for sophisticated mouse genome engineering with potential applications in other mammalian species.

Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes.

The CRISPR/Cas9 system has been employed to efficiently edit the genomes of diverse model organisms. CRISPR-mediated mouse genome editing is typically accomplished by microinjection of Cas9 DNA/RNA and single guide RNA (sgRNA) into zygotes to generate modified animals in one step. However, microinjection is a technically demanding, labor-intensive, and costly procedure with poor embryo viability. Here, we describe a simple and economic electroporation-based strategy to deliver Cas9/sgRNA ribonucleoproteins into mouse zygotes with 100% efficiency for in vivo genome editing. Our methodology, designated as CRISPR RNP Electroporation of Zygotes (CRISPR-EZ), enables highly efficient and high-throughput genome editing in vivo, with a significant improvement in embryo viability compared with microinjection. Using CRISPR-EZ, we generated a variety of editing schemes in mouse embryos, including indel (insertion/deletion) mutations, point mutations, large deletions, and small insertions. In a proof-of-principle experiment, we used CRISPR-EZ to target the tyrosinase (Tyr) gene, achieving 88% bi-allelic editing and 42% homology-directed repair-mediated precise sequence modification in live mice. Taken together, CRISPR-EZ is simple, economic, high throughput, and highly efficient with the potential to replace microinjection for in vivo genome editing in mice and possibly in other mammals.

Rescue of germline transmission from chimeras by IVF after sperm analysis.

Successful production of genetically modified mouse lines is dependent on germline transmission (GLT) of mutant alleles from chimeras. When natural mating fails to achieve GLT due to male infertility, sickness, or other problems, sperm can be harvested from chimeras and used for assisted reproductive technologies such as in vitro fertilization (IVF) to attempt to "rescue" GLT. However, a rational, evidence-based approach to determine if such extraordinary efforts should be attempted on a chimera has not been established. Therefore, in the present study we assessed the production, quality and genotype of epididymal sperm harvested from male chimeras generated by blastocyst or morula microinjection of gene targeted embryonic stem (ES) cell clones containing a LacZ expression cassette and that failed to achieve GLT. Results of this analysis enabled us to determine the cause of GLT failure, correlate coat color chimerism with the proportion of LacZ-positive sperm, and test the likelihood of achieving GLT by IVF. In 415 chimeras, 332 (80%) produced no offspring by natural mating ("infertile"), while 83 (20%) produced only wildtype offspring ("fertile"). Of the 332 infertile chimeras, 209 (63%) failed to produce any sperm whatsoever, 48 (15%) had extremely poor quality sperm, and 75 (23%) had good quality sperm. These results indicate that most chimeras that do not achieve GLT by natural mating are infertile, and the primary cause of infertility is failed spermatogenesis. Genotyping of sperm from 519 chimeras revealed a significant positive linear correlation between coat color chimerism and mean percentage of LacZ-positive sperm (R(2) = 0.95). Finally, IVF using good quality, LacZ-positive sperm from fertile and infertile chimeras "rescued" GLT for 19 out of 56 genes. We conclude that an assessment of coat color chimerism together with sperm quality and genotype can better inform the selection of chimeras for IVF to rescue GLT than coat color chimerism alone.

Conditional targeting of Ispd using paired Cas9 nickase and a single DNA template in mice.

CRISPR/Cas9 technology is a highly promising genome editing tool in the mouse, potentially overcoming the costs and time required for more traditional gene targeting methods in embryonic stem (ES) cells. Recently, compared to the wildtype nuclease, paired Cas9 nickase (Cas9n) combined with single guide RNA (sgRNA) molecules has been found to enhance the specificity of genome editing while reducing off-target effects. Paired Cas9n has been shown to be as efficient as Cas9 for generating insertion and deletion (indel) mutations by non-homologous end joining and targeted deletion in the genome. However, an efficient and reliable approach to the insertion of loxP sites flanking critical exon(s) to create a conditional allele of a target gene remains an elusive goal. In this study, we microinjected Cas9n RNA with sgRNAs together with a single DNA template encoding two loxP sites flanking (floxing) exon 2 of the isoprenoid synthase containing domain (Ispd) into the pronucleus and cytoplasm of C57BL/6NCr one-cell stage zygotes. After surgical transfer, one F0 mouse expressing a conditional allele was produced (at a frequency of ∼8% of live pups born). The floxed allele was transmitted through the germline to F1 progeny, and could be successfully recombined using Cre recombinase. This study indicates that conditional targeting can be accomplished effectively using paired Cas9n and a single DNA template.

Combining sperm plug genotyping and coat color chimerism predicts germline transmission.

There has been a significant increase in the use of C57BL/6N-derived ES cells for the production of gene knockout mice. However, the potential for germline transmission (GLT) from chimeras on this genetic background has been observed to be highly variable. Using coat color as an indicator of somatic chimerism to infer the extent of chimeric contribution to the germ cell population, even highly agouti C57BL/6N-derived chimeras can fail to achieve GLT. We investigated the extent to which quantitative PCR genotyping for a marker gene expressed in gene targeted ES cells can be performed on DNA extracted from sperm present in copulatory plugs to determine the contribution of ES cells to the germ cells. We found that an objective assessment of sperm DNA from copulatory plugs combined with a subjective assessment of coat color chimerism can be used to accurately inform the selection of chimeras for breeding that are likely to achieve GLT. These results indicate that, compared to random selection of chimeras, including an analysis of copulatory plugs to set chimeras for breeding can help to reduce costs, minimize time, and facilitate research for projects requiring the production, selection, breeding, and testing of chimeras to generate gene-targeted mice.

Rederivation of transgenic mice from iPS cells derived from frozen tissue.

In mice, induced pluripotent stem (iPS) cells with embryonic stem (ES)-like characteristics have been derived by ectopic expression of four transcription factors in somatic cells: Sox2, Oct3/4, Klf4 and/or c-Myc. To date, iPS cells have only be made from freshly harvested tissues and cells. However, if iPS cells could be derived from frozen tissues and cells, then cryopreservation of tissues such as mouse tails could conceivably become a reliable alternative to the more traditional formats, like germplasm and ES cells, for the archiving of genetically altered mouse lines. To test this hypothesis, we sought to demonstrate that a live transgenic mouse line could be recovered from transgenic iPS cells derived from cryopreserved mouse tissues. Tails and tail-derived fibroblasts from a DsRED transgenic mouse were cryopreserved in the presence of 5% dimethylsulfoxide (DMSO) in liquid nitrogen for 1 week and 1 month, respectively. Afterward, tissues and cells were thawed and underwent nuclear reprogramming by molecular transfection to derive iPS cells which generated germline confirmed transgenic mice. Our results demonstrate for the first time that iPS cells can be efficiently derived from frozen-stored-thawed tail tissue and fibroblasts and used to re-establish a transgenic mouse line. Therefore, this study provides conclusive evidence that, as a practical matter, frozen tails and fibroblasts can be used as an effective and reliable alternative to frozen germplasm and ES cells for the storage, maintenance, and distribution of genetically-altered mutant mice.