Proportion Of GFP-positive Cells Research Articles

Construction of a Mouse Model for Myeloproliferative Neoplasms and an Evaluation System

To construct a myeloproliferative neoplasms (MPN) transplanted mouse model with JAK2-V617F, MPLW515L or CALR-Type I gene mutation, and establish a systematic evaluation system to verify the success of model construction. The bone marrow c-kit+ cells of the mice were obtained by the following steps: The mice were killed by cervical dislocation, the femur, tibia and ilium were separated, and the bone marrow cells were collected. The c-kit+ cells were sorted after incubation with CD117 magnetic beads. The method of constructing mouse primary mutant cells is as follows: A gene mutation vector with a GFP tag was constructed by the retroviral system, and the retroviral vector was packaged into the Platinum-E cells to obtain the virus supernatant, and then used it to infect the c-kit+ cells of mice. The MPN mouse model was constructed as follows: the mouse primary c-kit+ cells containing the mutant genes were collected after infection, and then transplanted them via the tail vein into the female recipient mice of the same species which were irradiated with a lethal dose of gamma rays (8.0 Gy). The MPN mouse model was evaluated as follows: After transplantation, the peripheral blood of the mice was regularly collected from the tail vein to perform the complete blood count test, and the size of spleen and the degree of bone marrow fibrosis were estimated. The mouse c-kit+ cells with the mutant genes were successfully obtained from the bone marrow. MPN mouse model was successfully constructed: The peripheral blood cells of the MPN-transplanted mice carried exogenous implanted GFP-positive cells, and the white blood cells (WBC), platelet (PLT) and hematocrit (HCT) were all increased; the body weight loss, and the water and food intake were reduced in the transplanted mice; further pathological analysis showed that the transplanted mice displayed splenomegaly and bone marrow fibrosis. These results suggested that the MPN mouse model was successfully constructed. According to the common and different characteristics of the three MPN mouse model, a preliminary evaluation system for judging the success of MPN mouse model construction was summarized, which mainly included the following indicators, for example, the proportion of GFP-positive cells in the peripheral blood of mice; WBC, PLT and HCT; the degree of spleen enlargement and the bone marrow fibrosis. The MPN mouse model with JAK2-V617F, MPLW515L or CALR-Type I gene mutation is successfully established by retroviral system, which can provide an important experimental animal model for the research of MPN pathogenesis and drug-targeted therapy.

Integration of Cell-Penetrating Peptides with Rod-like Bionanoparticles: Virus-Inspired Gene-Silencing Technology.

Inspired by the high gene transfer efficiency of viral vectors and to avoid side effects, we present here a 1D rod-like gene-silencing vector based on a plant virus. By decorating the transacting activator of transduction (TAT) peptide on the exterior surface, the TAT-modified tobacco mosaic virus (TMV) achieves a tunable isoelectric point (from ∼3.5 to ∼9.6) depending on the TAT dose. In addition to enhanced cell internalization, this plant virus-based vector (TMV-TAT) acquired endo/lysosomal escape capacity without inducing lysosomal damage, resulting in both high efficiency and low cytotoxicity. By loading silencer green fluorescent protein (GFP) siRNA onto the TMV-TAT vector (siRNA@TMV-TAT) and interfering with GFP-expressing mouse epidermal stem cells (ESCs/GFP) in vitro, the proportion of GFP-positive cells could be knocked down to levels even lower than 15% at a concentration of ∼100% cell viability. Moreover, by interfering with GFP-expressing highly metastatic hepatocellular carcinoma (MHCC97-H/GFP) tumors in vivo, treatment with siRNA@TMV-TAT complexes for 10 days achieved a GFP-negative rate as high as 80.8%. This work combines the high efficiency of viral vectors and the safety of nonviral vectors and may provide a promising strategy for gene-silencing technology.

UTX Affects Neural Stem Cell Proliferation and Differentiation through PTEN Signaling.

SummaryNeural stem cell (NSC) proliferation and differentiation in the developing brain is a complex process precisely regulated by intrinsic and extrinsic signals. Although epigenetic modification has been reportedly involved in the regulation of the cerebral cortex, whether UTX, an H3K27me3 demethylase, regulates the development of cerebral cortex during the embryonic period is unclear. In this study, we demonstrate that Utx deficiency by knockdown and conditional knockout increases NSC proliferation and decreases terminal mitosis and neuronal differentiation. Furthermore, we find that impairment of cortical development caused by lack of Utx is less significant in males than in females. In addition, UTX demethylates H3K27me3 at the Pten promoter and promotes Pten expression. P-AKT and P-mTOR levels are increased in the Utx conditional knockout cortices. Finally, Utx or Pten overexpression can rescue the impairment of brain development caused by Utx loss. These findings may provide important clues toward deciphering brain diseases.

Migration of Mesenchymal Stem Cells of Bursal Tissue after Rotator Cuff Repair in Rats.

Purpose The purpose of this study is to verify migration of mesenchymal stem cells of bursal tissue into the healing site after rotator cuff repair in rats. Methods Fischer rats and green fluorescent protein (GFP)-transgenic rats were used. Bursal tissue from GFP rats was isolated and transplanted into tendon repair sites in Fischer rats. We examined the histology of the rotator cuff and the proportion of GFP-positive cells in the repaired rotator cuff 1, 3, and 6 weeks after surgery. Results Cell migration was observed during the third and sixth week after surgery. We also found mesenchymal stem cells and formed bursal cluster patterns in the repaired rotator cuff tendons. Conclusion Mesenchymal stem cells migrated from bursal tissue and infiltrated the repaired rotator cuff tendons. Clinical Relevance Mesenchymal stem cells from bursal tissue can contribute to the healing progress of the repaired rotator cuff.

GFP stable transfection facilitated the characterization of lung cancer stem cells.

Cancer stem cells (CSCs) are a subset of cancer cells that play key roles in metastasis and cancer relapse. The elimination of CSCs is very important during cancer therapy. To develop drugs that target CSCs, the isolation and identification of putative CSCs are required. Some of the characteristics of CSCs are assessed by cell survival assays. In such experiments, the density of the cells seeded on the plates may affect the experimental results, leading to potentially inaccurate conclusions. In this study, a new assay to facilitate the characterization of CSCs has been developed by stable transfection of GFP, using the A549 lung cancer cell line as a model. A putative CSC line, A549 sphere cells, was obtained by culturing A549 cells in ultra-low dishes in serum-free medium. To ensure that the putative CSCs were grown under the same conditions as the A549-GFP cells and were not affected by the number of cells seeded, A549 sphere cells were mixed with GFP stably transfected A549 (A549-GFP) cells. The mixture was subjected to flow cytometry assay and inverted fluorescence microscopy to detect changes in the proportion of GFP-positive cells after treatment. A549 sphere cells had a slower proliferation rate and an improved chemoresistance. They also showed differentiation ability. This work suggests that mixing GFP stably transfected cancer cells with putative CSCs may facilitate the identification of CSCs, making it convenient for studies of targeted CSCs.

Bone marrow-derived cells from the footprint infiltrate into the repaired rotator cuff

Cells from the bone marrow are considered important during the rotator cuff repair process, but the kinetics of bone marrow-derived cells in this process is unknown. To analyze the kinetics of bone marrow cells during the rotator cuff repair process, to review whether or not they are histologically involved in rotator cuff healing, and to analyze the biomechanics of the repaired tissues. Bone marrow chimeric rats that express green fluorescent protein (GFP) only in bone marrow- and circulation-derived cells were created. Bilateral supraspinatus tendons were separated from the greater tuberosity of the humeral head to produce a rotator cuff transection model. Drilling into the bone marrow was performed in the greater tuberosity of the right humerus and the supraspinatus tendon was repaired (drilling group), while the supraspinatus tendon was repaired on the left shoulder without drilling (control group). We examined the histology of the rotator cuff, the ultimate force-to-failure, and the proportion of GFP-positive cells in the repaired rotator cuff at 2, 4 and 8 weeks after surgery. Mesenchymal cells were observed in the repaired rotator cuff at 2 weeks in both groups. There were more GFP-positive cells in the drilling group than the control group at 2, 4 and 8 weeks. The ultimate force-to-failure was significantly higher in the drilling group than the control group at 4 and 8 weeks. Bone marrow-derived cells passed through holes drilled in the humerus footprint, infiltrated the repaired rotator cuff and contributed to postsurgical rotator cuff healing.

Abstract 618: Anti-tumor effects of Notch pathway activation in Gastrointestinal Stromal Tumors cells

Abstract Gastrointestinal Stromal Tumors (GISTs) are the most common sarcomas of the GI tract and harbor a gain-of-function mutation of the KIT receptor tyrosine kinase. The introduction of imatinib, a specific inhibitor of KIT, has significantly extended survival of many GIST patients. Unfortunately, patients inevitably develop resistance, prompting interest in understanding GIST biology and developing new therapeutic options. Notch signaling may act as an oncogene or a tumor suppressor depending cellular context. In neuroendocrine tumors Notch signaling acts constantly as a tumor suppressor. Additionally, histone deacetylase inhibitors (HDACi) were recently shown to induce Notch signaling in neuroendocrine neoplasms. Given that GIST is of neuroectodermal origin, we hypothesized that inhibition of growth in GIST cells by HDAC inhibitors is mediated by activation of Notch signaling. To characterize Notch signaling in GIST, we assessed the expression of Notch-1, and its effector human hairy enhancer of split-1 (HES1) in human GIST cell lines (GIST-T1 and GIST 882) by quantitative RT-PCR. To determine the activity of Notch signaling in GIST, we assayed the effect of Notch activation in GIST cells by transduction with retroviral vectors to overexpress active forms of Notch1 and HES1 in GIST cells. The proportion of GFP-positive cells in unselected culture was assessed to determine the transgene's effect on cell growth. To study the effect of HDAC inhibition, we quantified viability, caspase activity, cell cycle analysis and expression of Notch pathway genes after HDAC inhibition with suberoylanilide hydroxamic acid (SAHA). Notch signaling genes (Notch1, HES1) were weakly expressed at the basal level in cells compared to other sarcomas. Notch signaling with a constitutively active, truncated form of the receptor (ICN-1) and HES1 potently induced growth arrest in GIST cell lines. The proportion of ICN-1 GFP+ cells dramatically declined after transduction compared to GFP- cells, (decrease of 69% at day 7 and 78% at day 9 after transduction). Vector empty-transduced GFP+ cells gave no change in the proportion of positive cells. SAHA caused dose dependent decrease in viability, activation of caspase 3 and 7, increase of sub-G1 population (6 fold increase at 1µM, p<0.01), as well as upregulation of Notch-1 expression (5 fold at 1µM, p<0.01). Retroviral silencing of downstream targets of Notch (dnHes-1 and dnMastermind [dnMAM]) partially rescued GIST-T1 cells from the inhibitory effects of SAHA (61% v 105%, p<0.05 for dnHes-1 and 61 v 87%, p<0.01 for dnMAM after 2 days of treatment with SAHA 0.6 µM). These results suggest that Notch signaling is a tumor suppressor in GIST cells and the activation of Notch signaling by HDAC inhibitors may represent a new strategy for treating GIST patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 618. doi:10.1158/1538-7445.AM2011-618

99 EFFICIENCY OF CATIONIC LIPID-BASED DNA TRANSFECTION OF BOVINE IVF-DERIVED ZONA-FREE BLASTOCYSTS

We have previously reported that zona pellucida (ZP) removal and cationic lipid-based transfection (lipofection) of 1-cell stage bovine embryos with siRNA or DNA did not affect development to the blastocyst (BL) stage (Bertolini et al. 2006 Reprod. Fertil. Dev. 18, 168–169). Moreover, approximately half of green fluorescent protein (GFP)-transfected embryos showed various levels of fluorescence at the BL stage, which was also recently confirmed by others (O’Meara et al. 2010 Reprod. Fertil. Dev. 22, 224). The objective of this study was to evaluate the effect of the timing of ZP removal on the efficiency of lipofection in Day 8 bovine IVF-derived blastocyst stage embryos. In vitro-derived (IVP) embryos, produced based on (Bertolini et al. 2004 Reproduction 128, 341–354), were allocated to one of three experimental groups: (a) G-1, ZP removal on Day 1 of development, in embryos at the 1-cell stage (7 days before transfection); (b) G-2, ZP removal performed on Day 7 BL (24 h before transfection); and (c) G-3, naturally hatched Day 8 BL. On Day 8 of development, zona-free BL from G1, G2, and G3 were transfected by the incubation in 5 μL Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and 12 μg DNA (pEGFP, Clontech Laboratories, Mountain View, CA, USA) in 100 μL Opti-MEM I (Invitrogen), at 39°C for 30 to 60 min. Transfected embryos were cultured for 24 h, when qualitative and quantitative assessments of the transfection efficiency were done by fluorescence microscopy. Positively-transfected BL were DNA-stained for the estimation of the total GFP-positive cells and total cell numbers (ANOVA and Tukey’s test, P < 0.05), and for the estimation of the proportion of GFP-positive cells in each embryonic cell lineage (chi-square test; P < 0.05). Embryos showing at least one GFP-positive cell were qualitatively considered transfected. Cleavage rate on Day 2 (144/197, 73.1%) and BL rate on Day 7 (122/197, 61.9%) were similar between groups (chi-square test; P < 0.05). No qualitative differences in transfection were seen between groups (Table 1), with cell transfection being restricted to the embryonic trophectodermal (TE) cell lineage. Embryo developmental kinetics was slightly delayed in G-1, which also had lower proportion of GFP-positive cells per total TE cell number (GFP/TE) when compared with results with more developed G-2 and G-3 BL. In fact, the number and proportion of GFP-positive cells per embryo appeared to increase more in a stage-dependent manner, rather than due to the time intervals used for ZP removal (Day 1 or Day 7). In summary, lipofection was effective for the transfection of bovine BL, but at a rather low efficiency on an embryo basis. Such procedure may be useful in studies requiring the delivery of nucleic acids specific to the trophectodermal embryonic cell lineage. Table 1.Efficiency of cationic lipid-based DNA transfection of bovine IVF-derived zona-free blastocysts Supported by CNPq and FINEP/Brazil.

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

BackgroundMurine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone.MethodsA MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection.ResultsVectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene.ConclusionThese studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.

Evaluation of cationic liposomes composed of an amino acid–based lipid for neuronal transfection

We investigated the ability of cationic liposomes composed of 1,5-dihexadecyl N-arginyl-L-glutamate (Arg-Glu2C 16) to carry nucleic acids into neuronal cells. Such liposomes have been shown to have a remarkable capacity for transfecting immortalized cell lines. Lipoplexes between the Arg-Glu2C 16 liposomes and plasmid DNA encoding green fluorescent protein (GFP) were analyzed in terms of lipoplex formation, intracellular DNA trafficking, transfection efficiency, and cytotoxicity in neuronal SH-SY5Y cells. A maximum number of cells expressing GFP was obtained with lipoplexes at a lipid-to-DNA ratio of 15. With these lipoplexes, 16% of the cells were GFP-positive, which was approximately fourfold higher than the level obtained with a commercially available transfection reagent, Lipofectamine 2000. Furthermore, as a result of the low cytotoxicity of the Arg-Glu2C 16 lipoplexes, the proportion of GFP-positive cells could be increased to 25% by increasing the concentration of lipoplexes that was applied to the cells. We have demonstrated that Arg-Glu2C 16, as a model cationic amino acid–based lipid, has a high capability as a gene carrier, even for neuronal transfection. From the Clinical Editor In this study, specific cationic liposomes were characterized as nucleic acid transfection agents for neuronal cells. A fourfold higher transfection rate with low cytotoxicity was reported compared to Lipofectamine 2000, a commercial reagent. The authors conclude that the studied cationic liposomes have a high capability as a gene carrier for neuronal transfection. This may become clinically significant in future gene therapy efforts of neuronal diseases.

Paracrine factors of vascular endothelial cells facilitate cardiomyocyte differentiation of mouse embryonic stem cells

For myocardial regeneration therapy, the low differentiation capability of functional cardiomyocytes sufficient to replace the damaged myocardial tissue is one of the major difficulties. Using Nkx2.5-GFP knock-in ES cells, we show a new efficient method to obtain cardiomyocytes from embryonic stem (ES) cells. The proportion of GFP-positive cells was significantly increased when ES cells were cultured with a conditioned medium from aortic endothelial cells (ECs), accompanied by upregulation of cardiac-specific genes as well as other mesodermal genes. The promotion was more prominent when EC-conditioned medium was added at an early stage of ES cell differentiation culture (Day 0–3). Inhibitors of bone morphogenic protein (BMP), cyclooxygenase (COX), and nitric oxide synthetase (NO) prevented the promotion of cardiomyogenesis by EC-conditioned medium. These results suggest that supplementation of EC-conditioned medium enables cardiomyocytes to be obtained efficiently through promotion of mesoderm induction, which is regulated by BMP, COX, and NOS.

Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfection

Transfection with polyethylenimine (PEI) was evaluated as a method for the generation of recombinant Chinese hamster ovary (CHO DG44) cell lines by direct comparison with calcium phosphate-DNA coprecipitation (CaPO4) using both green fluorescent protein (GFP) and a monoclonal antibody as reporter proteins. Following transfection with a GFP expression vector, the proportion of GFP-positive cells as determined by flow cytometry was fourfold higher for the PEI transfection as compared to the CaPO4 transfection. However, the mean level of transient GFP expression for the cells with the highest level of fluorescence was twofold greater for the CaPO4 transfection. Fluorescence in situ hybridization on metaphase chromosomes from pools of cells grown under selective pressure demonstrated that plasmid integration always occurred at a single site regardless of the transfection method. Importantly, the copy number of integrated plasmids was measurably higher in cells transfected with CaPO4. The efficiency of recombinant cell line recovery under selective pressure was fivefold higher following PEI transfection, but the average specific productivity of a recombinant antibody was about twofold higher for the CaPO4-derived cell lines. Nevertheless, no difference between the two transfection methods was observed in terms of the stability of protein production. These results demonstrated the feasibility of generating recombinant CHO-derived cell lines by PEI transfection. However, this method appeared inferior to CaPO4 transfection with regard to the specific productivity of the recovered cell lines.

Targeting Wnt Signaling in Myeloma In Vivo; Differential Effects on Tumor Burden and Myeloma Bone Disease.

Multiple myeloma is characterized by uncontrolled proliferation of myeloma cells within the bone marrow and the development of a severe osteolytic bone disease. In addition to a well characterized increase in osteoclastic bone resorption, myeloma bone disease is associated with a reduction in bone formation. Osteoblast differentiation and bone formation are regulated in vivo by canonical Wnt signaling and activation of β-catenin. Therefore increasing Wnt signaling in the bone microenvironment in multiple myeloma may prevent the development of myeloma bone disease. In support of this, we have previously demonstrated that activation of Wnt signaling with lithium chloride (LiCl) in the 5TGM1 murine model of myeloma reduces tumor burden and osteolytic bone disease. However, we also found that LiCl treatment increased subcutaneous (s.c.) tumor growth. This suggests that the reduction in tumor burden within the bone microenvironment may be an indirect effect mediated through the effects of LiCl to prevent myeloma bone disease. The aim of the current study was to determine the effect of specific molecular blockade of Wnt signaling in myeloma cells in vivo. 5TGM1-GFP myeloma cells were transfected by electroporation with either myc-tagged dominant negative TCF4 (DNTCF4) or pcDNA. Following stable selection by culture in G418, expression of DNTCF4 was confirmed by western blot for myc. No difference was found in the growth rates of 5TGM1-pcDNA or 5TGM1-DNTCF4 in vitro. Treatment with LiCl or Wnt3A had no significant effect on cell viability in vitro, but significantly increased β-catenin activity, as measured by TOPFLASH activity in 5TGM1-pcDNA cells. This increase was not observed in 5TGM1-DNTCF4, confirming that expression of DNTCF4 blocked Wnt signaling induced by LiCl in 5TGM1 myeloma cells. C57Bl/KaLwRij mice were inoculated with 5TGM1-pcDNA or 5TGM1-DNTCF4 cells by either intravenous (i.v.) or s.c. injection. Mice were treated from time of tumor cell inoculation with 200mg/kg/day LiCl or vehicle control (d.H20) by oral gavage for 28 days. I.v. inoculation of myeloma cells resulted in a significant increase in serum IgG2bκ concentrations and the proportion of GFP-positive cells in the bone marrow. A significant reduction in trabecular bone volume was also observed. MicroCT analysis of the tibia demonstrated that LiCl significantly increased trabecular bone volume in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice. LiCl significantly decreased serum IgG2bκ concentrations in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice, with a greater effect in 5TGM1-DNTCF4 myeloma-bearing mice. FACS analysis of GFP-positive cells demonstrated that LiCl significantly reduced tumor burden in the bone marrow in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice. However, following s.c inoculation, LiCl significantly increased s.c. tumor volume of 5TGM1-pcDNA tumors, but had no effect on 5TGM1-DNTCF4 s.c. tumor volume. Taken together these results demonstrate that the effect of increasing Wnt signaling in myeloma is dependent upon the microenvironment. By specific inhibition of β-catenin activity in myeloma cells combined with systemic stimulation of the Wnt signaling pathway, our results suggest that increasing Wnt signaling in myeloma in vivo has dual effects; firstly to enhance myeloma growth directly, and secondly to enhance osteoblast differentiation and thus indirectly reduce tumor burden in bone, highlighting the importance of the bone marrow microenvironment in regulating myeloma growth and survival.

Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67-GFP knock-in mouse.

Gamma-aminobutyric acid (GABA)ergic neurons in the central nervous system regulate the activity of other neurons and play a crucial role in information processing. To assist an advance in the research of GABAergic neurons, here we produced two lines of glutamic acid decarboxylase-green fluorescence protein (GAD67-GFP) knock-in mouse. The distribution pattern of GFP-positive somata was the same as that of the GAD67 in situ hybridization signal in the central nervous system. We encountered neither any apparent ectopic GFP expression in GAD67-negative cells nor any apparent lack of GFP expression in GAD67-positive neurons in the two GAD67-GFP knock-in mouse lines. The timing of GFP expression also paralleled that of GAD67 expression. Hence, we constructed a map of GFP distribution in the knock-in mouse brain. Moreover, we used the knock-in mice to investigate the colocalization of GFP with NeuN, calretinin (CR), parvalbumin (PV), and somatostatin (SS) in the frontal motor cortex. The proportion of GFP-positive cells among NeuN-positive cells (neocortical neurons) was approximately 19.5%. All the CR-, PV-, and SS-positive cells appeared positive for GFP. The CR-, PV, and SS-positive cells emitted GFP fluorescence at various intensities characteristics to them. The proportions of CR-, PV-, and SS-positive cells among GFP-positive cells were 13.9%, 40.1%, and 23.4%, respectively. Thus, the three subtypes of GABAergic neurons accounted for 77.4% of the GFP-positive cells. They accounted for 6.5% in layer I. In accord with unidentified GFP-positive cells, many medium-sized spherical somata emitting intense GFP fluorescence were observed in layer I.