Bat-mouse bone marrow chimera: a novel animal model for dissecting the uniqueness of the bat immune system

Yong, Kylie Su Mei; Ng, Justin Han Jia; Her, Zhisheng; Hey, Ying Ying; Tan, Sue Yee; Tan, Wilson Wei Sheng; Irac, Sergio Erdal; Liu, Min; Chan, Xue Ying; Gunawan, Merry; Foo, Randy Jee Hiang; Low, Dolyce Hong Wen; Mendenhall, Ian Hewitt; Chionh, Yok Teng; Dutertre, Charles-Antoine; Chen, Qingfeng; Wang, Lin-Fa

Bats. Eonycteris spelaea (common name, cave nectar bat), our species of interest, was captured in Singapore at dusk using mist nets and transferred to clean customized bat bags for transportation. All animal processing work was conducted in accordance to with approved guidelines, methods and permits from Duke-NUS Medical School and SingHealth Experimental Medicine Centre (2015/SHS/1088). Bats were anaesthetised using isoflurane and exsanguinated via cardiac bleed. Various samples, such as, spleen and bone marrow were harvested. The spleen tissues were mashed through a 100 µm filter (Thermo Fisher scientific, USA) in DMEM medium (Thermo Fisher scientific, USA). The single-cell suspension was washed and re-suspended in media supplemented with 10% fetal bovine serum (FBS). Bone marrows were processed by flushing femurs and tibias with 5 mL of DMEM medium using 5 mL syringes (BD Biosciences, USA) and 27 gauge needles (BD Biosciences, USA). Contents flushed out of the bone marrow were mashed through a 100 µm filter in DMEM medium. The single-cell suspension was washed and re-suspended in media supplemented with 10% FBS. Cell viability was assessed using trypan blue. RNA isolation and quantitative polymerase chain reaction (qPCR). Blood samples were collected from NSG, C57BL/6, bat-mice and bats. RNA was prepared from these samples using RNeasy Micro kit (Qiagen, Netherlands). Reverse transcription was performed using iScript cDNA Synthesis Kit (BIO-RAD, USA) according to manufacturer's specifications. qPCR was subsequently performed in triplicates using SensiFAST ™ SYBR No-ROX Kit (Bioline, USA) and assays were run on the CFX96 Touch ™ Real-Time PCR Detection System (BIO-RAD, USA) under the following cycling condition: 95 °C for 5 minutes, followed by 40 cycles of 95 °C for 5 seconds and 58 °C for 30 seconds, and ending with a melt profile analysis. Bat-and mouse-specific qPCR primers were designed by first aligning the sequences from the two species, and subsequently targeting regions that are more than 50% (nucleotide) difference between the two species. Sequences of bat and mouse specific primers are listed in Supplementary Table 1. Immunization. Mice were immunized by injecting NP-KLH (Biosearch Technologies, USA) emulsified in IFA (Sigma-Aldrich, USA), intraperitoneally. To prepare emulsification, NP-KLH and IFA were added together and sonicated. One injection of 100 µg of NP-KLH was used to immunize the mice. After the first injection, mice were supplemented with 2 booster shots which were injected on the same day for 2 consecutive weeks. Mice were bled before immunization and bled again 2-weeks following the third injection for the detection of NP-specific antibodies.