Optogenetic control of early embryos labeling using photoactivatable Cre recombinase 3.0.

Establishing a highly efficient photoactivatable Cre recombinase PA-Cre3.0 can allow spatiotemporal control of Cre recombinase activity. This technique may help to elucidate cell lineages, as well as facilitate gene and cell function analysis during development. This study examined the blue light-mediated optical regulation of Cre-loxP recombination using PA-Cre3.0 transgenic early mouse pre-implantation embryos. We found that inducing PA-Cre3.0 expression in the heterozygous state did not show detectable recombination activation with blue light. Conversely, in homozygous embryos, DNA recombination by PA-Cre3.0 was successfully induced by blue light and resulted in the activation of the red fluorescent protein reporter gene, while almost no leaks of Cre recombination activity were detected in embryos without light illumination. Thus, we characterize the conditions under which the PA-Cre3.0 system functions efficiently in early mouse embryos. These results are expected to provide a new optogenetic tool for certain biological studies, such as developmental process analysis and lineage tracing in early mouse embryos.

Development of a tightly regulated copper-inducible transient gene expression system in Nicotiana benthamiana incorporating a suicide exon and Cre recombinase.

Chemical-inducible gene expression systems are commonly used to regulate gene expression for functional genomics in various plant species. However, a convenient system that can tightly regulate transgene expression in Nicotiana benthamiana is still lacking. In this study, we developed a tightly regulated copper-inducible system that can control transgene expression and conduct cell death assays in N. benthamiana. We tested several chemical-inducible systems using Agrobacterium-mediated transient expression and found that the copper-inducible system exhibited the least concerns regarding leakiness in N. benthamiana. Although the copper-inducible system can control the expression of some tested reporters, it is not sufficiently tight to regulate certain tested hypersensitive cell death responses. Using the MoClo-based synthetic biology approach, we incorporated the suicide exon HyP5SM/OsL5 and Cre/LoxP as additional regulatory elements to enhance the tightness of the regulation. This new design allowed us to tightly control the hypersensitive cell death induced by several tested leucine-rich repeat-containing proteins and their matching avirulence factors, and it can be easily applied to regulate the expression of other transgenes in transient expression assays. Our findings offer new approaches for both fundamental and translational studies in plant functional genomics.

Cell-specific expression of Cre recombinase in rat noradrenergic neurons via CRISPR-Cas9 system.

Noradrenergic neurons play a crucial role in the functioning of the nervous system. They formed compact small clusters in the central nervous system. To target noradrenergic neurons in combination with viral tracing and achieve cell-type specific functional manipulation using chemogenetic or optogenetic tools, new transgenic animal lines are needed, especially rat models for their advantages in large body size with facilitating easy operation, physiological parameter monitoring, and accommodating complex behavioral and cognitive studies. In this study, we successfully generated a transgenic rat strain capable of expressing Cre recombinase under the control of the dopamine beta-hydroxylase (DBH) gene promoter using the CRISPR-Cas9 system. Our validation process included co-immunostaining with Cre and DBH antibodies, confirming the specific expression of Cre recombinase. Furthermore, stereotaxic injection of a fluorescence-labeled AAV-DIO virus illustrated the precise Cre-loxP-mediated recombination activity in noradrenergic neurons within the locus coeruleus (LC). Through crossbreeding with the LSL-fluorescence reporter rat line, DBH-Cre rats proved instrumental in delineating the position and structure of noradrenergic neuron clusters A1, A2, A6 (LC), and A7 in rats. Additionally, our specific activation of the LC noradrenergic neurons showed effective behavioral readout using chemogenetics of this rat line. Our results underscore the effectiveness and specificity of Cre recombinase in noradrenergic neurons, serving as a robust tool for cell-type specific targeting of small-sized noradrenergic nuclei. This approach enhances our understanding of their anatomical, physiological, and pathological roles, contributing to a more profound comprehension of noradrenergic neuron function in the nervous system.

Colorimetric Barcoding to Track, Isolate, and Analyze Hematopoietic Stem Cell Clones.

In zebrafish, hematopoietic stem cells (HSCs) are born in the developing aorta during embryogenesis. From the definitive wave of hematopoiesis onward, blood homeostasis relies on self-renewal and differentiation of progeny of existing HSCs, or clones, rather than de novo generation. Here, we describe an approach to quantify the number and size of HSC clones at various times throughout the lifespan of the animal using a fluorescent, multicolor labeling strategy. The system is based on combining the multicolor Zebrabow system with an inducible, early lateral plate mesoderm and hematopoietic lineage specific cre driver (draculin (drl)). The cre driver can be temporally controlled and activated in early hematopoiesis to introduce a color barcoding unique to each HSC and subsequently inherited by their daughter cells. Clonal diversity and dominance can be investigated in normal development and blood disease progression, such as blood cancers. This adoptable method allows researchers to obtain quantitative insight into clonality-defining events and their contribution to adult hematopoiesis.

Spatiotemporal control of genome engineering in cone photoreceptors.

BACKGROUND: Cones are essential for color recognition, high resolution, and central vision; therefore cone death causes blindness. Understanding the pathophysiology of each cell type in the retina is key to developing therapies for retinal diseases. However, studying the biology of cone cells in the rod-dominant mammalian retina is particularly challenging. In this study, we used a bacterial artificial chromosome (BAC) recombineering method to knock in the "CreERT2" sequence into the Gnat2 and Arr3 genes, respectively and generated three novel inducible CreERT2 mice with different cone cell specificities. RESULTS: These models (Gnat2CreERT2, Arr3T2ACreERT2, and Arr3P2ACreERT2) express temporally controllable Cre recombinase that achieves conditional alleles in cone photoreceptors. Cre-LoxP recombination can be induced as early as postnatal day (PD) two upon tamoxifen injection at varying efficiencies, ranging from 10 to 15% in Gnat2CreERT2, 40% in Arr3T2ACreERT2, and 100% in Arr3P2ACreERT2. Notably, knocking in the P2A-CreERT2 cassette does not affect cone cell morphology and functionality. Most cone-phototransduction enzymes, including Opsins, CNGA3, etc. are not altered except for a reduction in the Arr3 transcript. CONCLUSIONS: The Arr3P2ACreERT2 mouse, an inducible cone-specific Cre driver, is a valuable line in studying cone cell biology, function, as well as its relationship with rod and other retinal cells. Moreover, the Cre activity can be induced by delivering tamoxifen intragastrically as early as PD2, which will be useful for studying retinal development or in rapid degenerative mouse models.

Highly selective transgene expression through the flip-excision switch system by using a unilateral spacer sequence.

The flip-excision switch (FLEX) system with an adeno-associated viral (AAV) vector allows expression of transgenes in specific cell populations having Cre recombinase. A significant issue with this system is non-specific expression of transgenes in tissues after vector injection. We show here that Cre-independent recombination events in the AAV genome carrying the FLEX sequence occur mainly during the production of viral vectors in packaging cells, which results in transgene expression in off-target populations. Introduction of a relatively longer nucleotide sequence between two recognition sites at the unilateral side of the transgene cassette, termed a unilateral spacer sequence (USS), is useful to suppress the recombination in the viral genome, leading to the protection of non-specific transgene expression with enhanced gene expression selectivity. Our FLEX/USS system offers a powerful strategy for highly specific Cre-dependent transgene expression, aiming at various applications for structural and functional analyses of target cell populations.

Development of an inducible excision system of a visual marker Ipomoea batatas Myb gene from the genome of transgenic cells.

In the plant genetic transformation process, single selection by a chemical-resistant marker gene occasionally allows the proliferation of non-transgenic cells, escaping selection pressure. The additional use of a visual marker gene is effective for accurate selection. For instance, R2R3-MYB genes are used for regulating anthocyanin biosynthesis; however, constitutive Myb expression in transgenic plants is not always desirable and may cause developmental abnormalities due to excess anthocyanin accumulation. To overcome the remaining problems in the use of Myb as a visible marker, we developed T-DNA. Ipomoea batatas Myb (IbMyb) and Cre expression cassettes were inserted between two loxP sequences, and the hygromycin phosphotransferase (HPT) and green fluorescent protein (GFP) expression cassettes were located outside of the loxP-IbMyb-Cre-loxP region. In the developed system, IbMyb and Cre were excised from the genomes of transgenic cells using heat-inducible Cre-loxP recombination. Upon heat treatment in a general incubator, green shoots emerged from purple tobacco transgenic calli that were pigmented with IbMyb expression. The excision of IbMyb from the genome of green shoots was confirmed using polymerase chain reaction (PCR) and sequencing. GFP expression was observed in the roots of the obtained green transgenic plants. We report that the system developed here operated successfully in tobacco, showing the potential to provide an easier and cheaper visual selection of transgenic cells in the genetic transformation process.

Catecholaminergic cell type-specific expression of Cre recombinase in knock-in transgenic rats generated by the Combi-CRISPR technology.

BACKGROUND: Cell groups containing catecholamines provide a useful model to study the molecular and cellular mechanisms underlying the morphogenesis, physiology, and pathology of the central nervous system. For this purpose, it is necessary to establish a system to induce catecholaminergic group-specific expression of Cre recombinase. Recently, we introduced a gene cassette encoding 2A peptide fused to Cre recombinase into the site between the C-terminus and translational termination codons of the rat tyrosine hydroxylase (TH) open reading frame by the Combi-CRISPR technology, which is a genomic editing method to enable an efficient knock-in (KI) of long DNA sequence into a target site. However, the expression patterns of the transgene and its function as well as the effect of the mutation on the biochemical and behavioral phenotypes in the KI strains have not been characterized yet. NEW METHOD: We aimed to evaluate the usefulness of TH-Cre KI rats as an experimental model for investigating the structure and function of catecholaminergic neurons in the brain. RESULTS: We detected cell type-specific expression of Cre recombinase and site-specific recombination activity in the representative catecholaminergic groups in the TH-Cre KI rat strains. In addition, we measured TH protein levels and catecholamine accumulation in the brain regions, as well as motor, reward-related, and anxiety-like behaviors, indicating that catecholamine metabolism and general behavior are apparently normal in these KI rats. CONCLUSIONS: TH-Cre KI rat strains produced by the Combi-CRISPR system offer a beneficial model to study the molecular and cellular mechanics for the morphogenesis, physiology, and pathology of catecholamine-containing neurons in the brain.

CRISPR RNA-guided integrase enables high-efficiency targeted genome engineering in Agrobacterium tumefaciens.

Agrobacterium tumefaciens, the causal agent of plant crown gall disease, has been widely used to genetically transform many plant species. The inter-kingdom gene transfer capability made Agrobacterium an essential tool and model system to study the mechanism of exporting and integrating a segment of bacterial DNA into the plant genome. However, many biological processes such as Agrobacterium-host recognition and interaction are still elusive. To accelerate the understanding of this important plant pathogen and further improve its capacity in plant genetic engineering, we adopted a CRISPR RNA-guided integrase system for Agrobacterium genome engineering. In this work, we demonstrate that INsertion of Transposable Elements by Guide RNA-Assisted TargEting (INTEGRATE) can efficiently generate DNA insertions to enable targeted gene knockouts. In addition, in conjunction with Cre-loxP recombination system, we achieved precise deletions of large DNA fragments. This work provides new genetic engineering strategies for Agrobacterium species and their gene functional analyses.

Transgenic mice encoding modern imaging probes: Properties and applications.

Modern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is the availability of animal models to track the molecules and cells in their natural environment in vivo. Owing to the integrity of the studied tissue and the high stability of transgene expression throughout life, transgenic mice encoding fluorescent proteins and biosensors represent unique tools for in vivo studies in norm and pathology. We review the strategies for targeting probe expression in specific tissues, cell subtypes, or cellular compartments. We describe the application of transgenic mice expressing fluorescent proteins for tracking protein expression patterns, apoptotic events, tissue differentiation and regeneration, neurogenesis, tumorigenesis, and cell fate mapping. We overview the possibilities of functional imaging of secondary messengers, neurotransmitters, and ion fluxes. Finally, we provide the rationale and perspectives for the use of transgenic imaging probes in translational research and drug discovery.

Postnatal Osterix but not DMP1 lineage cells significantly contribute to intramembranous ossification in three preclinical models of bone injury.

Murine models of long-bone fracture, stress fracture, and cortical defect are used to discern the cellular and molecular mediators of intramembranous and endochondral bone healing. Previous work has shown that Osterix (Osx+) and Dentin Matrix Protein-1 (DMP1+) lineage cells and their progeny contribute to injury-induced woven bone formation during femoral fracture, ulnar stress fracture, and tibial cortical defect repair. However, the contribution of pre-existing versus newly-derived Osx+ and DMP1+ lineage cells in these murine models of bone injury is unclear. We addressed this knowledge gap by using male and female 12-week-old, tamoxifen-inducible Osx Cre_ERT2 and DMP1 Cre_ERT2 mice harboring the Ai9 TdTomato reporter allele. To trace pre-existing Osx+ and DMP1+ lineage cells, tamoxifen (TMX: 100 mg/kg gavage) was given in a pulse manner (three doses, 4 weeks before injury), while to label pre-existing and newly-derived lineage Osx+ and DMP1+ cells, TMX was first given 2 weeks before injury and continuously (twice weekly) throughout healing. TdTomato positive (TdT+) cell area and cell fraction were quantified from frozen histological sections of injured and uninjured contralateral samples at times corresponding with active woven bone formation in each model. We found that in uninjured cortical bone tissue, Osx Cre_ERT2 was more efficient than DMP1 Cre_ERT2 at labeling the periosteal and endosteal surfaces, as well as intracortical osteocytes. Pulse-labeling revealed that pre-existing Osx+ lineage and their progeny, but not pre-existing DMP1+ lineage cells and their progeny, significantly contributed to woven bone formation in all three injury models. In particular, these pre-existing Osx+ lineage cells mainly lined new woven bone surfaces and became embedded as osteocytes. In contrast, with continuous dosing, both Osx+ and DMP1+ lineage cells and their progeny contributed to intramembranous woven bone formation, with higher TdT+ tissue area and cell fraction in Osx+ lineage versus DMP1+ lineage calluses (femoral fracture and ulnar stress fracture). Similarly, Osx+ and DMP1+ lineage cells and their progeny significantly contributed to endochondral callus regions with continuous dosing only, with higher TdT+ chondrocyte fraction in Osx+ versus DMP1+ cell lineages. In summary, pre-existing Osx+ but not DMP1+ lineage cells and their progeny make up a significant amount of woven bone cells (particularly osteocytes) across three preclinical models of bone injury. Therefore, Osx+ cell lineage modulation may prove to be an effective therapy to enhance bone regeneration.

Efficient ssODN-Mediated Targeting by Avoiding Cellular Inhibitory RNAs through Precomplexed CRISPR-Cas9/sgRNA Ribonucleoprotein.

Combined with CRISPR-Cas9 technology and single-stranded oligodeoxynucleotides (ssODNs), specific single-nucleotide alterations can be introduced into a targeted genomic locus in induced pluripotent stem cells (iPSCs); however, ssODN knockin frequency is low compared with deletion induction. Although several Cas9 transduction methods have been reported, the biochemical behavior of CRISPR-Cas9 nuclease in mammalian cells is yet to be explored. Here, we investigated intrinsic cellular factors that affect Cas9 cleavage activity in vitro. We found that intracellular RNA, but not DNA or protein fractions, inhibits Cas9 from binding to single guide RNA (sgRNA) and reduces the enzymatic activity. To prevent this, precomplexing Cas9 and sgRNA before delivery into cells can lead to higher genome editing activity compared with Cas9 overexpression approaches. By optimizing electroporation parameters of precomplexed ribonucleoprotein and ssODN, we achieved efficiencies of single-nucleotide correction as high as 70% and loxP insertion up to 40%. Finally, we could replace the HLA-C1 allele with the C2 allele to generate histocompatibility leukocyte antigen custom-edited iPSCs.

Chromosomal Rearrangements of Synthetic Yeast by SCRaMbLE.

Diversified genomes derived from chromosomal rearrangements are valuable materials for evolution. Naturally, chromosomal rearrangements occur at extremely low frequency to ensure genome stability. In the synthetic yeast genome project (Sc2.0), an inducible chromosome rearrangement system named Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is built to produce chromosomal rearrangements such as deletion, duplication, inversion, and translocation at high efficiency. Here, we detail the method to activate SCRaMbLE in a synthetic strain, to analyze the SCRaMbLEd genome, and to dissect the causative rearrangements for a desired phenotype after SCRaMbLEing.

Influence of specifically knocking out AMP-activated protein kinase α1 subunit gene in excitatory neurons in brain energy metabolism and cognitive function in mice / 中华神经医学杂志

Objective:To investigate the changes of brain energy metabolism and cognitive function in mice with specifically knocking out AMP-activated protein kinase α1 subunit ( AMPKα1) gene in the excitatory neurons by Cre-loxP recombination system. Methods:Sixteen 6-month-old mice with genotype AMPKα1 flox/flox/Camk2a-Cre/ERT2 obtained by hybrid breeding were randomly divided into AMPKα1 knockout group ( n=8) and AMPKα1 wild-type group ( n=8). Mice in the AMPKα1 knockout group were intraperitoneally injected 0.1 mL tamoxifen (20 mg/mL, dissolved in corn oil) daily for a consecutive 5 d to control AMPKα1 gene knockout in the excitatory neurons; and mice in the AMPKα1 wild-type group were intraperitoneally injected 0.1 mL corn oil daily for a consecutive 5 d. Seven d after that, Morris water maze and T maze experiments were employed to detect the spatial learning and memory abilities and spatial working memory of these mice; chemical exchange saturation transfer imaging (CEST) was used to observe the glucose metabolism in the hippocampus and cortex surrounding the hippocampus; Western blotting was used to detect the AMPKα1 and glutamate receptor 1 (GluR1) protein expressions in the hippocampus and cortex surrounding hippocampus of two groups. Results:(1) Morris water maze showed that, as compared with those in the AMPKα1 wild-type group, mice in the AMPKα1 knockout group had significantly prolonged escape latency ([13.90±3.72] s vs. [22.40±6.28] s; [11.95±3.86] s vs. [22.39±9.77] s]) on the 3 rd and 4 th d of experiment, statistically decreased times crossing the platform ([5.25±1.83] times vs. [1.75±1.28] times, P<0.05). (2) T-maze experiment showed that as compared with that of the AMPKα1 wild-type group, the free alternation rate in mice of the AMPKα1 knockout group was significantly decreased ([73.21±9.16]% vs. [48.21±11.29]%, P<0.05). (3) CEST showed that the glucose metabolism levels in the hippocampus and cortex surrounding the hippocampus of AMPKα1 knockout group were significantly lower than those in AMPKα1 wild-type group (1.51±0.81 vs. 2.77±0.67; 1.31±0.83 vs. 2.42±0.95, P<0.05). (4) Western blotting showed that the AMPKα1 and GluR1 protein expressions in the hippocampus and cortex surrounding the hippocampus of the AMPKα1 wild-type group were significantly higher than those of the AMPKα1 knockout group (AMPKα1: 0.70±0.05 vs. 0.49±0.03, 0.98±0.04 vs. 0.64±0.06; GluR1: 1.22±0.18 vs. 0.60±0.11, 0.96±0.08 vs. 0.79±0.04, P<0.05). Conclusion:Specifically knocking out AMPKα1 in excitatory neurons can result in abnormal glucose metabolism in the brain of mice, and thus cause cognitive dysfunction, whose mechanism may be related to excitatory synaptic disorder caused by energy metabolism disorder.

An AND-Gated Drug and Photoactivatable Cre-loxP System for Spatiotemporal Control in Cell-Based Therapeutics.

While engineered chimeric antigen receptor (CAR) T cells have shown promise in detecting and eradicating cancer cells within patients, it remains difficult to identify a set of truly cancer-specific CAR-targeting cell surface antigens to prevent potentially fatal on-target off-tumor toxicity against other healthy tissues within the body. To help address this issue, we present a novel tamoxifen-gated photoactivatable split-Cre recombinase optogenetic system, called TamPA-Cre, that features high spatiotemporal control to limit CAR T cell activity to the tumor site. We created and optimized a novel genetic AND gate switch by integrating the features of tamoxifen-dependent nuclear localization and blue-light-inducible heterodimerization of Magnet protein domains (nMag, pMag) into split Cre recombinase. By fusing the cytosol-localizing mutant estrogen receptor ligand binding domain (ERT2) to the N-terminal half of split Cre(2-59aa)-nMag, the TamPA-Cre protein ERT2-CreN-nMag is physically separated from its nuclear-localized binding partner, NLS-pMag-CreC(60-343aa). Without tamoxifen to drive nuclear localization of ERT2-CreN-nMag, the typically high background of the photoactivation system was significantly suppressed. Upon blue light stimulation following tamoxifen treatment, the TamPA-Cre system exhibits sensitivity to low intensity, short durations of blue light exposure to induce robust Cre-loxP recombination efficiency. We finally demonstrate that this TamPA-Cre system can be applied to specifically control localized CAR expression and subsequently T cell activation. As such, we posit that CAR T cell activity can be confined to a solid tumor site by applying an external stimulus, with high precision of control in both space and time, such as light.

Rational construction of genome-reduced and high-efficient industrial Streptomyces chassis based on multiple comparative genomic approaches.

BACKGROUND: Streptomyces chattanoogensis L10 is the industrial producer of natamycin and has been proved a highly efficient host for diverse natural products. It has an enormous potential to be developed as a versatile cell factory for production of heterologous secondary metabolites. Here we developed a genome-reduced industrial Streptomyces chassis by rational 'design-build-test' pipeline. RESULTS: To identify candidate large non-essential genomic regions accurately and design large deletion rationally, we performed genome analyses of S. chattanoogensis L10 by multiple computational approaches, optimized Cre/loxP recombination system for high-efficient large deletion and constructed a series of universal suicide plasmids for rapid loxP or loxP mutant sites inserting into genome. Subsequently, two genome-streamlined mutants, designated S. chattanoogensis L320 and L321, were rationally constructed by depletion of 1.3 Mb and 0.7 Mb non-essential genomic regions, respectively. Furthermore, several biological performances like growth cycle, secondary metabolite profile, hyphae morphological engineering, intracellular energy (ATP) and reducing power (NADPH/NADP+) levels, transformation efficiency, genetic stability, productivity of heterologous proteins and secondary metabolite were systematically evaluated. Finally, our results revealed that L321 could serve as an efficient chassis for the production of polyketides. CONCLUSIONS: Here we developed the combined strategy of multiple computational approaches and site-specific recombination system to rationally construct genome-reduced Streptomyces hosts with high efficiency. Moreover, a genome-reduced industrial Streptomyces chassis S. chattanoogensis L321 was rationally constructed by the strategy, and the chassis exhibited several emergent and excellent performances for heterologous expression of secondary metabolite. The strategy could be widely applied in other Streptomyces to generate miscellaneous and versatile chassis with minimized genome. These chassis can not only serve as cell factories for high-efficient production of valuable polyketides, but also will provide great support for the upgrade of microbial pharmaceutical industry and drug discovery.

Protocols for Studies on Genetically Engineered Mouse Models in Prostate Cancer.

Cancer studies have entered an era that is heavily focused on the contribution of the tumor microenvironment. For this reason, in vivo experimentation in an immunodeficient model system is no longer fit for purpose. As a consequence, numerous genetically engineered mouse models (GEMMs) which self-develop tumors have been developed to allow experiments to be performed in a fully immunocompetent setting. One of the most commonly used technologies is Cre-loxP recombination due to its unique ability to control target gene expression in a specified tissue type. However, the major limitation of these models remains the inability to generate sufficient numbers of age-matched mice for a synchronized experimental start date. For this reason, the derivation of cell lines from genetically modified murine prostate tissue is desirable and allows for the generation of syngeneic models via subcutaneous or orthotopic injection.

Targeted knock-in of CreER T2 in zebrafish using CRISPR/Cas9.

New genome-editing approaches, such as the CRISPR/Cas system, have opened up great opportunities to insert or delete genes at targeted loci and have revolutionized genetics in model organisms like the zebrafish. The Cre-loxp recombination system is widely used to activate or inactivate genes with high spatial and temporal specificity. Using a CRISPR/Cas9-mediated knock-in strategy, we inserted a zebrafish codon-optimized CreER T2 transgene at the otx2 gene locus to generate a conditional Cre-driver line. We chose otx2 as it is a patterning gene of the anterior neural plate that is expressed during early development. By knocking in CreER T2 upstream of the endogenous ATG of otx2, we utilized this gene's native promoter and enhancer elements to perfectly match CreER T2 and endogenous otx2 expression patterns. Next, by combining this novel driver line with a Cre-dependent reporter line, we show that only in the presence of tamoxifen can efficient Cre-loxp-mediated recombination be achieved in the anterior neural plate-derived tissues like the telencephalon, the eye and the optic tectum. Our results imply that the otx2:CreER T2 transgenic fish will be a valuable tool for lineage tracing and conditional mutant studies in larval and adult zebrafish.

Construction and identification of conditional islet βcell DEPTOR knockout mice / 实用医学杂志

Objective To study the function of Deptor gene on the regulation of diabetes mellitus in suc-cessfully constructed and identified islet β-cell conditionally DEPTOR knockout mice model. Method By cross-breeding Deptorflox/floxmice with Cre mice expressed conditional specifically in pancreatic β-cell,Deptorflox/+Cre+/-mice were acquired and their genotypic identification was then performed. As the mice model of this study, Deptorflox/floxCre+/-mice were generated by crossing Deptorflox/+Cre+/-mice with Deptorflox/floxmice.Genotypic identifica-tion was performed by PCR at the age of 3 weeks. Tamoxifen was administered through intraperitoneal injection to induce the activation of the Cre recombination in islet beta cells of 8 weeks mice.Double immunofluorescence label-ing was then applied to identify the knockout effect of DEPTOR gene. Results Ten Islets Deptor knockout mice models were successfully acquired after 10-month cross-breeding. Validated genotype by PCR analysis were Deptorflox/floxCre+/- and double immunofluorescence labeling showed a significant difference between knockout mice and rodent controls. Conclusion Our study successfully constructs the islets conditionally Deptor deleted mice model by using Cre-loxp recombination system,providing a promising appliable animal model for study of dia-betes mellitus pathogenetic mechanism.

Establishment of transgenic mice model with overexpression of neuritin in bone marrow / 实用医学杂志

Objective To establish transgenic mice model with over expression of neuritin in bone mar-row,for the further study on the function of neuritin protein in the treatment of peripheral neuropathy. Methods Two pairs of transgenic mice(loxp-stop-loxp-neuritin and lyz-Cre/+)were fed and propagated,the DNA from the mice tails extracted and the genotype of transgenic mice identified by PCR. The wild type mice with B6 were as-signed as the controls,and the immunofluorescence was used to detect the accuracy of the neuritinloxp/+ _lyz -Cre/+. Results The two trensgenetic homozygous mice had the ability to reproduce,and the hybrid offsprings were neuritinloxp/+_lyz-Cre/+,neuritinloxp/-_lyz-Cre/+,neuritinloxp/+_lyz-Cre/-,neuritinloxp/-_lyz-Cre/-. The re-sults were met with the Mendel′s law. The results of immunofluorescence showed that the expression of neuritin of neuritinloxp/+_lyz-Cre/+ mice in bone marrow was significantly higher than the wild type mice(P < 0.05). Con-clusion The PCR method is of high reliability for identification of sub pus genotype and the female neuritinloxp/+mice mating with the male lyz-Cre/+ ones is an effective way for obtaining the neuritinloxp/+_lyz-Cre/+ mice.