Optimizing dissection, sample collection and cell isolation protocols for frugivorous bats

Abstract

1. Bats are becoming increasingly important as an experimental model due to their unique biological features. These include the ability of powered flight, minimal consequences from a heightened metabolic state, extended longevity in most species and minimal inflammation in response to most otherwise pathogenic viruses. To date there has been limited work done on the optimal procedures for necropsy, extraction of tissues or preparation of cell suspensions for downstream experimental work. 2. Here we use Pteropus alecto black flying fox as an example model of the fruit bat to develop optimal procedures for anaesthetizing, necropsy methods, safety, sequence and protocols for cell/tissue extraction and isolation protocols. 3. These methods were successfully used to yield high-quality RNA, DNA and protein samples from tissues along with viable cells for various molecular and immunological studies. Procedures utilized are suitable for comparative biology studies with most protocols being directly modified from those used in mice and humans. While mainly optimized for the larger fruit bats (flying foxes) in this study, the majority of protocols can readily be adapted to all species of bats. 4. This study provides the framework for greater consistency with in vivo bat experiments, application for comparative biology studies and greater engagement of the bat community for suitable protocols to be harmoniously adopted.

Unique Evolution of Antiviral Tetherin in Bats

“…To compare the expression of alternative isoforms of P. alecto tetherin and the impact of treatment with immune-stimulating compounds, bats were treated with PBS, LPS (InvivoGen, San Diego, CA), or PIC (InvivoGen) as described previously ( 47 ). Briefly, 5 h post-intraperitoneal injection, bats were anesthetized and culled and organs were processed for RNA, DNA, protein, and cell suspensions as described previously ( 80 ). RNA libraries were prepared using RiboZero Plus rRNA-depletion kits (Illumina, San Diego, CA) and cDNA was generated using a mix of oligo-dT/random hexamer primers, prior to sequencing in 2×150PE on the Illumina HiSeq platform.…”

Interferon Regulatory Factors IRF1 and IRF7 Directly Regulate Gene Expression in Bats in Response to Viral Infection

“…Prior to processing, bats were transferred to a temperature-controlled facility, settled, euthanised and culled immediately by cardiac bleed to prevent any systemic effects from anesthesia. Processing of bats and the generation of PaKi cell lines was has been described previously ( Crameri et al., 2009 ; Irving et al., 2019 ). PaKiT03 (CVCL_DR89) and variants, Hek293T (CVCL_0063) and MdKi ( Liang et al., 2015 ) cell lines were all cultured in DMEM (GIBCO) with 10% (v/v) FBS.…”

Unique Evolution of Antiviral Tetherin in Bats

“…To compare the expression of alternative isoforms of P. alecto tetherin and the impact of treatment with immune-stimulating compounds, bats were treated with PBS, LPS (Invivogen, #tlrl-pb5lps) or poly(I:C) (Invivogen, #vac-pic) as published previously (46). Briefly, 5 h post-intraperitoneal injection, bats were anaesthetised, culled and organs were processed for RNA, DNA, protein and cell suspensions as described previously (78). RNA libraries were prepared using RiboZero Plus rRNA-depletion kits (Illumina, USA) and cDNA was generated using a mix of oligo-dT/random hexamer primers, prior to sequencing in 2×150PE on the Illumina HiSeq platform.…”