Transgenic overexpression of PLSCR1 using Rosa26 locus targeted conditional knock-in strategy in mice

Sonoor Majid1, Ashley Hernandez-gutierrez2, Dongqin Yang2, Chun Geun Lee2, M.D. Ph.D, Jack A. Elias2,3, M.D., Yang Zhou2, Ph.D.,

1Department of Biochemistry and Molecular Biology, University of Nebraska-Lincoln2Department of Molecular Microbiology and Immunology, Brown University

3Department of Internal Medicine, Warren Alpert Medical School, Brown University

Research sponsored by the National Heart, Lung, and Blood Institute, National Institutes of Health, R25HL088992

Phospholipid scramblase 1 (Plscr1) is the most studied member of the phospholipidscramblase protein family whose main function is the bidirectional and non-specifictranslocation of phospholipids between the inner and outer leaflets of the plasmamembrane. Activation of Plscr1 allows for the externalization of phosphatidylserine(PS) in the outer membrane, which acts as a dock for many biological processesincluding coagulation, apoptosis, and activation. Our previous data demonstrated thatnull mutations of murine Plscr1 augment lung Type 2 immune responses. Wehypothesize that Plscr1 is a potent inhibitor of innate immunity, and Type 2 immuneresponses would be diminished in Plscr1 overexpression animals. The aim of thisstudy is to generate Plscr1 overexpression (Plscr1 OE) mice using a newly developedRosa26 locus targeted conditional knock-in strategy. Specifically, LysM Cre mice werecrossed with Plscr1 OE mice to allow spontaneous overexpression of Plscr1 inmyeloid cells. In addition, Cre- ERT2 (estrogen receptor T2) mice were crossed withPlscr1 OE mice, and tamoxifen was administered to induce Plscr1 expression. Wecharacterized Plscr1 expression in lungs and livers of these animals using RT-PCR,Western blots, and Immunohistochemistry. These mice will be used in future studiesto determine the role of Plscr1 in Type 2 immune responses.

ABSTRACT

BACKGROUND & HYPOTHESIS

• LysM Cre mice crossing with Plscr1 OE mice allows spontaneous overexpression ofPlscr1 in myeloid cells in the livers and lungs of mice.

• Tamoxifen was able to induce Plscr1 expression in the lungs and livers of Plscr1 OEmice when crossed with Cre- ERT2 (estrogen receptor T2) mice.

METHODS• Chemicals: Tamoxifen was manufactured and supplied by Sigma-Aldrich.• Injection: Mice were injected with 320uL of Tamoxifen for 5 consecutive days. 48 hours after the

final injection, mice were sacrificed.• Immunoblot analysis: Following sacrifice, liver and lungs were lysed with RIPA buffer. Protein

content was quantified with the BCA method. Equal amounts of protein were loaded onto 4-20%acrylamide gels and transferred to a PVDF membrane. Membranes were incubated in primary andsecondary antibody and developed using an ECL chemiluminescence detection kit.

• Histology: Lung and liver tissue samples were loaded into cassettes, stored in 70% ethanol solutionand were sent to the Brown University of Pathology and Laboratory Medicine at 70 Ship Street,Providence, R.I. for slide preparation of cross sections for staining.

• Immunohistochemistry: Slides were dipped 3X Xylene, 100%, 90%, 80%, and 70% EtOH atdifferent duration. Slides were placed in 250mLAntigen Retrieval Buffer 1X. (abcam: ab93678).Slides were stained for paraffin sections following dehydration of samples. DAB PeroxidaseSubstrate Kit (SK-4100) afterwards to produce proper tissue staining for 1-3 minutes. Subsequently,Vector Nuclear Fast Red Counterstain (H-3405) was performed to obtain optimal stain intensity.

• Real Time-PCR: RNA was isolated from both Liver and Lung tissues using Trizol. cDNA wassynthesized from 0.13ug of total RNA using the iScript cDNA synthesis kit (Bio-Rad). iTaq UniversalSYBR Green Supermix (Bio-Rad) was mixed with cDNA and gene specific primers at a final volumeof 20μL. RT-PCR was performed using a CFX96 real time machine (Bio-Rad). All reactions wereperformed at least in duplicate and data was normalized to the expression of RpI13A. Fold changevalues were calculated using the following formula: 2-(target gene-housekeeping gene).

• These mice will be used in future studies to determine the role of Plscr1 in Type 2immune responses in OVA and HDM models in vivo, and role of Plscr1 in ILC2activation in vitro.

1. Sahu, S. K., Gummadi, S. N., Manoj, N., & Aradhyam, G. K. (2007). Phospholipid scramblases: an overview. Archives of biochemistry and biophysics, 462(1), 103-114.

2. Bevers, E. M., & Williamson, P. L. (2010). Phospholipid scramblase: an update. FEBS letters, 584(13), 2724-2730.

3. Kodigepalli, K. M., Bowers, K., Sharp, A., & Nanjundan, M. (2015). Roles and regulation of phospholipid scramblases. FEBS letters, 589(1), 3-14.

CONCLUSIONS

FUTURE DIRECTIONS

CITATIONS

ACKNOWLEDGEMENTS

RESULTS

• Phospholipid scramblase 1 (Plscr1) is the most studied member of the phospholipidscramblase protein family whose main function is the bidirectional and non-specifictranslocation of phospholipids between the inner and outer leaflets of the plasmamembrane1,2,3.

• Our previous data demonstrated that null mutations of murine Plscr1 augment lungType 2 immune responses.

• Hypothesis: We hypothesize that Plscr1 is a potent inhibitor of innate immunity, andType 2 immune responses would be diminished in Plscr1 overexpression in animals.

• The aim of this study is to generate Plscr1 overexpression (Plscr1 OE) mice using anewly developed Rosa26 locus targeted conditional knock-in strategy.

2) RT-PCR

3) Immunoblot analysis & Densitometry Analysis

4) Immunohistochemistry

LysM+Cre- Tamoxifen + Cre-Liver

LungLysM+Cre- Tamoxifen + Cre-Tamoxifen + ERT+

• We are greatly appreciative of support provided by The Leadership Alliance SummerResearch - Early Identification Program.

• I would like to thank Dr. Yang Zhou’s Molecular Microbiology and Immunology Lab forguidance and support in the conduct of this research.

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Figure 2. Results show relative mRNA expression of Plscr1 primers. Values were obtained for N=2.

1) Rosa-Plscr1 KI construct and breeding strategy

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Figure 3. Western blots and densitometry analysis of Plscr1 protein expression in lungs and livers.

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Figure 4. Plscr1 IHC in lungs and livers

Figure 1. Illustration of the knock-in strategy for breeding mice. The two gene expressions are LysM Cre mice crossedwith Plscr1 OE mice and Cre/ERT2 mice crossed with Plscr1 OE mice.

LoxP-Terminator-LoxP Plscr1

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