Comparison of Thermal and Mechanical Pain Testing Modalities in Sprague Dawley and Fischer 344 Rats ().

While rodents are used extensively for studying pain, there is a lack of reported direct comparisons of thermal and mechanical pain testing methods in rats of different genetic backgrounds. Understanding the range of interindividual variability of withdrawal thresholds and thermal latencies based on these testing methods and/or genetic background is important for appropriate experimental design. Testing was performed in two common rat genetic backgrounds: outbred Sprague-Dawley (SD) and inbred Fischer 344 (F344).

Improving laboratory animal genetic reporting: LAG-R guidelines.

The biomedical research community addresses reproducibility challenges in animal studies through standardized nomenclature, improved experimental design, transparent reporting, data sharing, and centralized repositories. The ARRIVE guidelines outline documentation standards for laboratory animals in experiments, but genetic information is often incomplete. To remedy this, we propose the Laboratory Animal Genetic Reporting (LAG-R) framework.

CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus (AAV) and 2-Cell Embryo Electroporation.

Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair templates and CRISPR-Cas reagents is the most common delivery method into embryos. However, this type of micromanipulation necessitates access to specialized equipment, is laborious, and requires a certain level of technical skill.

Comparison of Thermal and Mechanical Pain Testing Modalities in Sprague Dawley and Fischer 344 Rats ().

While rodents are used extensively for studying pain, there is a lack of reported direct comparisons of thermal and mechanical pain testing methods in rats of different genetic backgrounds. Understanding the range of interindividual variability of withdrawal thresholds and thermal latencies based on these testing methods and/or genetic background is important for appropriate experimental design. Testing was performed in two common rat genetic backgrounds: outbred Sprague-Dawley (SD) and inbred Fischer 344 (F344).

Role of nucleus accumbens D1-type medium spiny neurons in the expression and extinction of sign-tracking.

While sign-tracking, also known as autoshaping, has been studied for many decades, only recently has the tendency to show sign-tracking behavior been linked to the development and persistence of addiction. Sign-tracking is dependent upon dopamine activity in the nucleus accumbens (NAc). The NAc is comprised predominantly of medium spiny projection neurons (MSN) that can be differentiated by their D1-like or D2-like dopamine receptor expression.

Efficient DNA knock-in using AAV-mediated delivery with 2-cell embryo CRISPR-Cas9 electroporation.

Recent advances in CRISPR-Cas genome editing technology have been instrumental in improving the efficiency to produce genetically modified animal models. In this study we have combined four very promising approaches to come up with a highly effective pipeline to produce knock-in mouse and rat models. The four combined methods include: AAV-mediated DNA delivery, single-stranded DNA donor templates, 2-cell embryo modification, and CRISPR-Cas ribonucleoprotein (RNP) electroporation.

Rat Embryonic Stem Cell Transgenesis.

The availability of reliable germline competent rat embryonic stem cell (ESC) lines that can be genetically manipulated provides an important tool for generating new rat models. Here we describe the process for culturing rat ESCs, microinjecting the ESCs into rat blastocysts, and transferring the embryos to surrogate dams by either surgical or non-surgical embryo transfer techniques to produce chimeric animals with the potential to pass on the genetic modification to their offspring.

Gene Targeting in Rat Embryonic Stem Cells.

Rat germline-competent embryonic stem (ES) cell lines have been available since 2008, and rat models with targeted mutations have been successfully generated using ES cell-based genome targeting technology. This chapter will focus on the procedures of gene targeting in rat ES cells.

Electroporation-Mediated CRISPR/Cas9 Genome Editing in Rat Zygotes.

Genetic engineering in the rat has been revolutionized by the development of CRISPR-based genome editing tools. Conventional methods for inserting genome editing elements such as CRISPR/Cas9 reagents into rat zygotes include cytoplasmic or pronuclear microinjections. These techniques are labor-intensive, require specialized micromanipulator equipment, and are technically challenging.

KSOM-R supports both mouse and rat preimplantation embryo development in vitro.

A modified KSOM for rat embryo culture (KSOM-R), which has enriched taurine, glycine, glutamic acid, and alanine, promoted rat embryo development in vitro. Since mice and rats share similar amino acid profiles in their female reproductive tracts, this study explored whether KSOM-R would also have a positive effect on mouse embryo development and if KSOM-R modifications could extend its shelf time at 2-8 °C for consistency. We first examined the effects of newly made (≤1 month at 2-8 °C) antibiotics-free KSOM-R (mKSOM-R), antibiotics-free KSOM (mKSOM) and KSOM on the development of in vivo or in vitro derived C57BL/6NJ zygotes.

Celsr1 suppresses Wnt5a-mediated chemoattraction to prevent incorrect rostral migration of facial branchiomotor neurons.

In the developing hindbrain, facial branchiomotor (FBM) neurons migrate caudally from rhombomere 4 (r4) to r6 to establish the circuit that drives jaw movements. Although the mechanisms regulating initiation of FBM neuron migration are well defined, those regulating directionality are not. In mutants lacking the Wnt/planar cell polarity (PCP) component Celsr1, many FBM neurons inappropriately migrate rostrally into r3.

The Ighmbp2D564N mouse model is the first SMARD1 model to demonstrate respiratory defects.

Spinal muscular atrophy with respiratory distress type I (SMARD1) is a neurodegenerative disease defined by respiratory distress, muscle atrophy and sensory and autonomic nervous system defects. SMARD1 is a result of mutations within the IGHMBP2 gene. We have generated six Ighmbp2 mouse models based on patient-derived mutations that result in SMARD1 and/or Charcot-Marie Tooth Type 2 (CMT2S).

Molecular and culture-based assessment of the microbiome in a zebrafish (Danio rerio) housing system during set-up and equilibration.

Background: Zebrafish used in research settings are often housed in recirculating aquaculture systems (RAS) which rely on the system microbiome, typically enriched in a biofiltration substrate, to remove the harmful ammonia generated by fish via oxidation. Commercial RAS must be allowed to equilibrate following installation, before fish can be introduced. There is little information available regarding the bacterial community structure in commercial zebrafish housing systems, or the time-point at which the system or biofilter reaches a microbiological equilibrium in RAS in general.

Mutational analyses of novel rat models with targeted modifications in inflammatory bowel disease susceptibility genes.

Mutations and single base pair polymorphisms in various genes have been associated with increased susceptibility to inflammatory bowel disease (IBD). We have created a series of rat strains carrying targeted genetic alterations within three IBD susceptibility genes: Nod2, Atg16l1, and Il23r, using CRISPR/Cas9 genome editing technology. Knock-out alleles and alleles with known human susceptibility polymorphisms were generated on three different genetic backgrounds: Fischer, Lewis and Sprague Dawley.

The gene: characterization of wild type, knock-in, and knock-out phenotypes in rats.

is a ubiquitous autophagy gene responsible, in part, for formation of the double-membrane bound autophagosome that delivers unwanted cellular debris and intracellular pathogens to the lysosome for degradation. A single, nonsynonymous adenine to guanine polymorphism resulting in a threonine to alanine amino acid substitution (T300A) directly preceded by a caspase cleavage site (DxxD) causes an increased susceptibility to Crohn's disease (CD) in humans. The mechanism behind this increased susceptibility is still being elucidated, however, the amino acid change caused by this point mutation results in increased ATG16L1 protein sensitivity to caspase 3-mediated cleavage.

A Nonsurgical Embryo Transfer Technique for Fresh and Cultured Blastocysts in Rats.

The use of a nonsurgical embryo transfer technique in rodents eliminates the potential pain, distress, and health complications that may result from a surgical procedure and as such, represents a refinement in rodent assisted reproductive techniques. A nonsurgical technique has not been previously developed for use with rat embryos. Here we describe an efficient method to deliver either fresh or cultured blastocyst stage embryos to the uterine horn of pseudopregnant female rats using a rat nonsurgical embryo transfer (rNSET) device.

Media optimization to promote rat embryonic development to the blastocyst stage in vitro.

Efficient model production in rats that incorporates newly developed genetic editing and embryo transfer tools, such as CRISPR/Cas9 technology and non-surgical embryo transfer, requires availability of an optimal embryo culture system. However, current technologies for in vitro manipulation of rat gametes, including embryo culture techniques, are less advanced compared to those in mice. In this study, we (1) identified a culture medium that was able to support optimal rat embryonic development by comparing two rat culture media: mR1ECM (modified rat 1-cell embryo culture medium) and KSOM-R (modified potassium simplex optimized medium for rats), and (2) evaluated the effect of glutamine dipeptides: alanyl-l-glutamine and glycyl-l-glutamine, on rat embryonic development.

Using Vaginal Impedance Measurement to Identify Proestrus in Rats Given Luteinizing Hormone Releasing Hormone (LHRH) Agonist.

Vaginal cytology is the most common method of monitoring the estrous cycle in rats; however, this test requires specific technical training and can be subject to interpretation. Vaginal impedance offers a quicker and less technically challenging alternative and has been used successfully to identify estrus in normally cycling breeder rats. We hypothesize that vaginal impedance can also be used to stage the estrous cycle in rats that have been given luteinizing hormone releasing hormone (LHRH) for timed mating.

Development of a novel severe mouse model of spinal muscular atrophy with respiratory distress type 1: FVB-nmd.

Spinal Muscular Atrophy with Respiratory Distress type 1 (SMARD1) is an autosomal recessive disease that develops early during infancy. The gene responsible for disease development is immunoglobulin helicase μ-binding protein 2 (IGHMBP2). IGHMBP2 is a ubiquitously expressed gene but its mutation results in the loss of alpha-motor neurons and subsequent muscle atrophy initially of distal muscles.

A novel conditional ZsGreen-expressing transgenic reporter rat strain for validating Cre recombinase expression.

The Cre/loxP recombination system has revolutionized the ability to genetically manipulate animal genomes in order to conditionally control gene expression. With recent advances in genome editing, barriers to manipulating the rat genome have been overcome and it is now possible to generate new rat strains (Cre drivers) in which Cre recombinase expression is carefully controlled temporally and/or spatially. However, the ability to evaluate and characterize these Cre driver strains is limited by the availability of reliable reporter rat strains.

Versatile cell ablation tools and their applications to study loss of cell functions.

Targeted cell ablation is a powerful approach for studying the role of specific cell populations in a variety of organotypic functions, including cell differentiation, and organ generation and regeneration. Emerging tools for permanently or conditionally ablating targeted cell populations and transiently inhibiting neuronal activities exhibit a diversity of application and utility. Each tool has distinct features, and none can be universally applied to study different cell types in various tissue compartments.

Rat Genome and Model Resources.

Rats remain a major model for studying disease mechanisms and discovery, validation, and testing of new compounds to improve human health. The rat's value continues to grow as indicated by the more than 1.4 million publications (second to human) at PubMed documenting important discoveries using this model.

Swine models, genomic tools and services to enhance our understanding of human health and diseases.

The pig is becoming increasingly important as a biomedical model. Given the similarities between pigs and humans, a greater understanding of the underlying biology of human health and diseases may come from the pig rather than from classical rodent models. With an increasing need for swine models, it is essential that the genomic tools, models and services be readily available to the scientific community.

Derivation of Transgene-Free Rat Induced Pluripotent Stem Cells Approximating the Quality of Embryonic Stem Cells.

Although a variety of reprogramming strategies have been reported to create transgene-free induced pluripotent stem (iPS) cells from differentiated cell sources, a fundamental question still remains: Can we generate safe iPS cells that have the full spectrum of features of corresponding embryonic stem (ES) cells? Studies in transgene-free mouse iPS cells have indicated a positive answer to this question. However, the reality is that no other species have a derived transgene-free iPS cell line that can truly mimic ES cell quality. Specifically, critical data for chimera formation and germline transmission are generally lacking.