This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. A link to any extended data will be provided when the final version is posted online. Research Article: Methods/New Tools | Novel Tools and Methods Targeting microglia using Cx3cr1-cre lines: revisiting the specificity Xiao-Feng Zhao1, Mahabub Maraj Alam1, Tingting Huang1, Xinjun Zhu2 and Yunfei Huang1 1Department of Neuroscience and Experimental Therapeutics, Albany Medical College, Albany, NY 12208, USA 2Department of Molecular and Cellular Physiology; The IBD Center, Division of Gastroenterology, Department of Medicine, Albany Medical College, Albany, NY 12208, USA https://doi.org/10.1523/ENEURO.0114-19.2019 Received: 18 March 2019 Revised: 11 May 2019 Accepted: 28 May 2019 Published: 14 June 2019 X. Zhao and Y.H. designed research; X. Zhao performed research; X. Zhao, M.M.A., T.H., and X. Zhu contributed unpublished reagents/analytic tools; X. Zhao analyzed data; X. Zhao and Y.H. wrote the paper. Funding: HHS | NIH | National Institute of Neurological Disorders and Stroke (NINDS) NS093045 ; Funding: HHS | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) DK099566 . Conflict of Interest: Authors report no conflict of interest. This work was supported by the NIH (grant NS093045 to Y.H). Correspondence should be addressed to Yunfei Huang at [email protected]. Cite as: eNeuro 2019; 10.1523/ENEURO.0114-19.2019 Alerts: Sign up at www.eneuro.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2019 Zhao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 Manuscript Title Page 2 1. Manuscript Title: 3 Targeting microglia using Cx3cr1-cre lines: revisiting the specificity 4 5 2. Abbreviated Title 6 Specificity of microglia Cx3cr1-cre lines 7 8 3. Author Names and Affiliations 9 1) Xiao-Feng Zhao - Department of Neuroscience and Experimental Therapeutics, 10 Albany Medical College, Albany, NY 12208, USA 11 2) Mahabub Maraj Alam - Department of Neuroscience and Experimental Therapeutics, 12 Albany Medical College, Albany, NY 12208, USA 13 3) Tingting Huang - Department of Neuroscience and Experimental Therapeutics, 14 Albany Medical College, Albany, NY 12208, USA 15 4) Xinjun Zhu - Department of Molecular and Cellular Physiology, Albany Medical 16 College, Albany, NY 12208, USA; The IBD Center, Division of Gastroenterology, 17 Department of Medicine, Albany Medical College, Albany, NY 12208, USA 18 5) Yunfei Huang - Department of Neuroscience and Experimental Therapeutics, Albany 19 Medical College, Albany, NY 12208, USA 20 21 4. Author Contributions 22 X. Zhao and Y. Huang designed research; X. Zhao performed research; T. Huang 23 provided assistance on animal handling; X. Zhao and Y. Huang analyzed data and 24 wrote the paper; M. M. Alam and X. Zhu provided helpful advice and edited the 25 manuscript 26 27 5. Correspondence should be addressed to 28 Yunfei Huang -Department of Neuroscience and Experimental Therapeutics, Albany 29 Medical College, Albany, NY 12208, USA 30 E-mail: [email protected] 31 32 1 33 6. Numbers of Figures: 4 34 7. Number of Tables: 0 35 8. Number of Multimedia: 0 36 9. Number of words or Abstract: 235 37 10. Number of words for Significance Statement: 118 38 11. Number of words for Introduction: 513 39 12. Number of words for Discussion: 1313 40 41 13. Acknowledgements: 42 43 14. Conflict of Interest: 44 Authors report no conflict of interest 45 46 15. Funding sources: This work was supported by the NIH (grant NS093045 to Y.H). 47 2 48 Abstract: 49 50 Microglia play a pivotal role in maintaining homeostasis of the CNS. There is growing interest in 51 understanding how microglia influence normal brain function and disease progression. Several 52 microglia-specific Cx3cr1-Cre lines have been developed and have become indispensable tools 53 in many investigations of microglial function. However, some recent studies have reported that 54 these lines may have significant leakage into neurons. Other studies have reported that Cx3cr1 is 55 expressed in non-microglial cells, including neurons and astrocytes, in vitro or in vivo either 56 during brain development or upon neurological insult. All these reports raise serious concerns 57 about the trustworthiness of these Cre-lines and whether the conclusions drawn from previous 58 studies are valid. Here, we found that a floxed fluorescent reporter mouse line which has been 59 frequently used to verify Cre lines displayed spontaneous expression of the GFP reporter, 60 independent of Cre recombinase, thus revealing a potential caveat in assessing cre lines. We 61 further confirmed that two Cx3cr1-Cre mouse lines can drive fluorescent reporter expression 62 largely restrictively in microglia. Finally, we clarified that these two mouse lines maintain 63 microglia-specific expression even following excitatory injury. Together, our findings confirm 64 that two previously created Cx3cr1-Cre lines remain as invaluable tools for studying microglia. 65 Moreover, to ensure the quality of data generated and the soundness of conclusions drawn from 66 such data, it should be compulsory to thoroughly examine reporter lines for spontaneous 67 leakiness when labeling cells to study CNS function and diseases. 68 69 70 3 71 Significance Statement: 72 Microglia-specific Cre-lines are essential for studying the role of microglia in the CNS. Several 73 Cx3cr1-Cre lines have been developed and used in a number of landmark studies. However, 74 there is growing concern in the microglia research community regarding potential leakiness of 75 Cre-lines into neurons. The conclusions drawn from previous studies are also being questioned 76 and key ongoing studies have been stalled. We found that a GFP-reporter mouse lines used in a 77 previous study displays spontaneous leakiness into neurons, independent of Cre recombinase. 78 Furthermore, we confirmed that two Cre-lines are microglia-specific and thus can be redeployed 79 without hesitation. Our study also suggests that testing for potential leakiness of GFP-reporter 80 lines should be included as a control in cell-tracing experiments. 81 4 82 83 Introduction: 84 85 Microglia are present nearly uniformly throughout the entire central nervous system, where they 86 constantly prune synapses and repair minor injuries. Microglia are also the chief resident 87 immune cells, part of the innate immune response to ‘danger’ signals. Genetic tools that 88 specifically target microglia are essential for unlocking the roles of microglia in the CNS and 89 neurological diseases. 90 91 Endogenous Cx3cr1 is a selective marker for microglia in the CNS, whereas its ligand 92 fractalkine is expressed in neurons. Several mouse lines have been created for studying microglia 93 (Wieghofer and Prinz, 2016). The Cx3cr1-GFP reporter line (Stock #005582, Jackson 94 laboratory) was created by replacing the coding part of the Cx3cr1 gene with green fluorescent 95 protein (GFP) (Jung et al., 2000). This reporter line has been widely used to label microglia for 96 visualization. The expression pattern of Cx3cr1-driven GFP reporter is specific to microglia over 97 the entire period of postnatal brain development and appears to be maintained even following an 98 excitatory injury (Kim et al., 2015). Using a BAC transgenic strategy, two constitutive and two 99 inducible Cx3cr1 promoter-driving cre lines were created (Gong et al., 2007; Gong et al., 2003; 100 Parkhurst et al., 2013; Peng et al., 2016; Yona et al., 2013). The constitutive Cx3cr1-cre line 101 created by Jung et al. (Stock #025524, Jackson laboratory) was recently reported to have 102 significant leakage into neurons (Haimon et al., 2018; Zhang et al., 2018). Another constitutive 103 Cx3cr1-Cre line was developed by the Mutant Mouse Resource and Research Center (MMRRC). 104 This line was reported to drive microglial-specific expression of a reporter (also created by 105 MMRRC) in the majority of animals examined (Hwang et al., 2017). However, about 10% of the 5 106 animals displayed significant leakage into neurons (Hwang et al., 2017). In another study, this 107 line was reported to drive the expression of mT/mG reporter (Stock # 007676, Jackson 108 Laboratory) selectively in microglia (Zhao et al., 2018). Two Cx3cr1-CreERT2 inducible lines 109 were created separately by two laboratories. In initial studies, these lines were found to deliver 110 microglial-specific expression (Parkhurst et al., 2013; Yona et al., 2013). However, a recent 111 study found that one of these inducible lines displays significant leakage of the Rosa-Green 112 reporter (Stock # 007906, Jackson laboratory) into neurons (Zhang et al., 2018). It is unclear why 113 the leakage into neurons varies among the different Cx3cr1-cre lines and studies (Hwang et al., 114 2017; Parkhurst et al., 2013; Zhang et al., 2018; Zhao et al., 2018), but the problem has raised 115 concerns about whether there is a reliable cre-line that targets microglia with good specificity. 116 Moreover, investigators now hesitate to use these lines and some have questioned the validity of 117 the data generated in the previous studies (Hwang et al., 2017; Parkhurst et al., 2013; Zhang et 118 al., 2018; Zhao et al., 2018). Lack of validated cre lines that specifically target microglia would 119 be a significant setback to the field. Therefore, it is imperative to understand how the variable 120 leakiness among these lines is occurring and how it might be rectified. Here, we performed a 121 thorough characterization of three popular GFP reporter lines, along with two Cx3cr1 promoter- 122 driven Cre lines.