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. Moreover, microinjection techniques often result in lower embryo survival due to the mechanical stress on the embryo. In this protocol, we developed an optimized method to deliver large DNA repair templates to work in conjunction with CRISPR-Cas9 genome editing without the need for microinjection. This protocol combines AAV-mediated DNA delivery of single-stranded DNA donor templates along with the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) by electroporation to modify 2-cell embryos. Using this novel strategy, we have successfully produced targeted knock-in rat models carrying insertion of DNA sequences from 1.2 to 3.0 kb in size with efficiencies between 42% and 90%.

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

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). Male and female, 10- to 14-wk-old F344 and SD rats were used to assess withdrawal thresholds in 3 different modalities: the Randall-Selitto test (RST), Hargreaves test (HT), and tail flick test (TFT). The RST was performed by using an operator-controlled handheld instrument to generate a noxious pressure stimulus to the left hind paw. The HT and the TFT used an electronically controlled light source to deliver a noxious thermal stimulus to the left hind paw or tail tip, respectively. Rats of each sex and genetic background underwent one type of test on day 0 and day 7. Withdrawal thresholds and thermal latencies were compared among tests. No significant differences were observed. Our findings can serve as a guide for researchers considering these nociceptive tests for their experiments.

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. Here we determined how reducing activity of D1-type MSNs in the NAc affects the expression and extinction of sign-tracking. To address this, we transfected the NAc of transgenic male and female rats that selectively express Cre recombinase in D1-type MSNs with a DIO viral vector expressing hM4Di. Cre- rats were given the same viral infusion but did not express the hM4Di receptor and therefore served as controls. Rats were then conditioned to associate lever presentations with pellet delivery. After sign-tracking was established, all rats were administered clozapine-n-oxide (CNO) prior to three additional conditioning sessions to assess the effects of NAc D1-MSNs inhibition on sign-tracking in the presence of reward. CNO treatment did not alter the expression of sign-tracking in Cre+ or Cre- rats. Next rats underwent extinction training where lever presentations occurred without pellet delivery and all rats received a CNO injection prior to each extinction session. In these extinction conditions, Cre+ rats exhibited robust extinction of sign-tracking across sessions, whereas Cre- rats did not. To determine if D1-MSN inhibition merely produced a temporary cessation of sign-tracking or instead had facilitated a persistent loss of sign-tracking, we evaluated the reemergence of sign-tracking in a test for reconditioning. During testing, reintroduction of the CS-US pairing did not promote the reemergence of sign-tracking in Cre+ rats, but restored sign-tracking in Cre- rats. Thus, chemogenetic inhibition of NAc D1-MSNs promoted extinction of sign-tracking. Collectively, these data suggest that D1-MSNs play an important role in resistance to extinction that typifies sign-tracking behavior.

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. Using this new combined approach, we were able to produce successfully targeted knock-in rat models containing either Cre or Flp recombinase sequences with knock-in efficiencies over 90%. Furthermore, we were able to produce a knock-in mouse model containing a Cre recombinase targeted insertion with over 50% knock-in efficiency directly comparing efficiencies to other commonly used approaches. Our modified AAV-mediated DNA delivery with 2-cell embryo CRISPR-Cas9 RNP electroporation technique has proven to be highly effective for generating both knock-in mouse and knock-in rat models.

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. Here, we describe a simple and effective method for zygote electroporation in which CRISPR/Cas9 reagents are introduced into rat zygotes via pores produced by precise electrical pulses applied to the cells. Zygote electroporation allows for high-throughput efficient genome editing in rat embryos.

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.

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.

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. We then investigated the effect of extended shelf life (6 months at 2-8 °C) of mKSOM-R and mKOSM on the development of C57BL/6NJ mouse and Sprague Dawley (SD) rat embryos. The results showed that there were no significant differences in cleavage, blastocyst, and hatching rates of C57BL/6NJ embryos among the three freshly made media. After 6 months of storage at 2-8 °C, mKSOM-R and mKSOM were still able to support the development of in vivo C57BL/6NJ zygotes at comparable rates seen with newly made (≤1 month at 2-8 °C) KSOM (control) in terms of cleavage, blastocyst formation and hatching. There were also no significant differences in total cell numbers in day 4 blastocysts among the three groups. After surgical embryo transfers, C57BL/6NJ blastocysts cultured in mKSOM-R (6 months at 2-8 °C) and newly made (≤1 month at 2-8 °C) KSOM culture developed into live pups. These pups had no gross abnormalities in animal morphology and growth. SD zygotes cultured in mKSOM-R stored at 2-8 °C for 6 months developed at comparable rates in cleavage, blastocyst and hatching rates when compared to those cultured in newly made mKSOM-R (≤1 month at 2-8 °C). The data showed that, although no significant beneficial effects were observed on mouse embryo development, mKSOM-R was able to support both mouse and rat embryo development in vitro. Additionally, mKSOM-R and mKSOM can be stored at 2-8 °C for at least 6 months without significantly compromising quality. This study suggests that it is possible to reduce the media inventory by using only mKSOM-R to culture both mouse and rat embryos, and quality media with extended shelf life can be made through modifications.

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. We hypothesized that Celsr1 normally blocks inappropriate rostral migration of FBM neurons by suppressing chemoattraction towards Wnt5a in r3 and successfully tested this model. First, FBM neurons in Celsr1; Wnt5a double mutant embryos never migrated rostrally, indicating that inappropriate rostral migration in Celsr1 mutants results from Wnt5a-mediated chemoattraction, which is suppressed in wild-type embryos. Second, FBM neurons migrated rostrally toward Wnt5a-coated beads placed in r3 of wild-type hindbrain explants, suggesting that excess Wnt5a chemoattractant can overcome endogenous Celsr1-mediated suppression. Third, rostral migration of FBM neurons was greatly enhanced in Celsr1 mutants overexpressing Wnt5a in r3. These results reveal a novel role for a Wnt/PCP component in regulating neuronal migration through suppression of chemoattraction.

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). Here we describe the characterization of one of these models, Ighmbp2D564N (human D565N). The Ighmbp2D564N/D564N mouse model mimics important aspects of the SMARD1 disease phenotype, including motor neuron degeneration and muscle atrophy. Ighmbp2D564N/D564N is the first SMARD1 mouse model to demonstrate respiratory defects based on quantified plethysmography analyses. SMARD1 disease phenotypes, including the respiratory defects, are significantly diminished by intracerebroventricular (ICV) injection of ssAAV9-IGHMBP2 and the extent of phenotypic restoration is dose-dependent. Collectively, this model provides important biological insight into SMARD1 disease development.

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. METHODS: A zebrafish housing system was monitored at multiple different system sites including tank water in six different tanks, pre- and post-particulate filter water, the fluidized bed biofilter substrate, post-carbon filter water, and water leaving the ultra-violet (UV) disinfection unit and entering the tanks. All of these samples were collected in quadruplicate, from prior to population of the system with zebrafish through 18 weeks post-population, and analyzed using both 16S rRNA amplicon sequencing and culture using multiple agars and annotation of isolates via matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry. Sequencing data were analyzed using traditional methods, network analyses of longitudinal data, and integration of culture and sequence data. RESULTS: The water microbiome, dominated by Cutibacterium and Staphylococcus spp., reached a relatively stable richness and composition by approximately three to four weeks post-population, but continued to evolve in composition throughout the study duration. The microbiomes of the fluidized bed biofilter and water leaving the UV disinfection unit were distinct from water at all other sites. Core taxa detected using molecular methods comprised 36 amplicon sequence variants, 15 of which represented Proteobacteria including multiple members of the families Burkholderiaceae and Sphingomonadaceae. Culture-based screening yielded 36 distinct isolates, and showed moderate agreement with sequencing data. CONCLUSIONS: The microbiome of commercial RAS used for research zebrafish reaches a relatively stable state by four weeks post-population and would be expected to be suitable for experimental use following that time-point.

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 availability of these rat models will contribute to our understanding of the basic biological roles of these three genes as well as provide new potential IBD animal models.

The Atg16l1 gene: characterization of wild type, knock-in, and knock-out phenotypes in rats.

ATG16L1 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. To generate novel rat strains carrying genetic alterations in the rat Atg16l1 gene, we first characterized the wild-type rat gene. We identified four alternative splice variants with tissue-specific expression. Using CRISPR-Cas9 genome editing technology, we developed a knock-in rat model for the human ATG16L1 T300A CD risk polymorphism, as well as a knock-out rat model to evaluate the role of Atg16l1 in autophagy as well as its potential effect on CD susceptibility. These are the first reported rat strains with alterations of the Atg16l1 gene. Consistent with studies of the effects of human ATG16L1 polymorphisms, models exhibit morphological abnormalities in both Paneth and goblet cells, but do not develop spontaneous intestinal permeability or inflammatory bowel disease. Analysis of the gut microbiota does not show inherent differences in bacterial composition between wild-type and genetically modified animals. These Atg16l1 strains are valuable new animal models for the study of both autophagy and CD susceptibility.

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. The rNSET device is composed of a Teflon catheter and a hub that attaches to a 2 µL pipette. Oxytocin is used to dilate the cervix before the delivery of blastocysts, allowing passage of the rNSET catheter directly into the uterine horn for embryo delivery. The efficiency of recovery of pups after nonsurgical embryo transfer is similar to the efficiency after surgical embryo transfer. Furthermore, the technique is not stressful to the subjects, as demonstrated by the absence of a decrease in weight or increase in fecal corticosterone level in recipients of embryos delivered nonsurgically, without the use of anesthesia or analgesia.

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. We also investigated the possibility of simplifying the KSOM-R culture procedure by increasing the volume of culture medium, reducing the need for daily medium changes. The results showed that rat embryos cultured in KSOM-R developed faster than those cultured in mR1ECM. Both alanyl-l-glutamine and glycyl-l-glutamine showed detrimental effects on rat embryonic development when supplemented in KSOM-R at the same concentration as glutamine. By increasing the volume of KSOM-R, rat zygotes were able to develop without daily medium refreshment at a similar rate and developmental competence as those in smaller volumes with daily medium changes. These results represent important improvements to rat embryo culture methods and will assist in more efficient production of rat models.

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. Vaginal impedance measurements and vaginal cytology were performed in LHRH-primed female rats (n = 36) at the expected peak of proestrus and paired with proven stud males. Breeding success was determined by gross necropsy to detect embryo implantation sites in the female rats. We found that the predictive rates of vaginal cytology and impedance measurement for proestrus were similar; however, both methods resulted in high proportions of false positive and false negative determinations (28% and 31%, respectively). We further hypothesized that females respond to LHRH at variable rates, resulting in variable times of peak proestrus. To test this, vaginal impedance measurements were performed multiple times throughout the expected day of proestrus in LHRH-primed female rats (n = 36). Females were either paired with a male 24 h after reaching the proestrus threshold (n = 18) or paired according to our standard protocol at 1300 h on the day after the expected proestrus (n = 18). Sequential measurements reduced false positive and negative rates (14% and 8%, respectively). Pregnancy rates did not differ based on the time of pairing during expected estrus. Overall, we determined vaginal impedance can be more successful than vaginal cytology at identifying proestrus in the rat, but only if multiple measurements are taken.

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. The current SMARD1 mouse model arose from a spontaneous mutation originally referred to as neuromuscular degeneration (nmd). The nmd mice have the C57BL/6 genetic background and contain an A to G mutation in intron 4 of the endogenous Ighmbp2 gene. This mutation causes aberrant splicing, resulting in only 20-25% of full-length functional protein. Several congenital conditions including hydrocephalus are common in the C57BL/6 background, consistent with our previous observations when developing a gene therapy approach for SMARD1. Additionally, a modifier allele exists on chromosome 13 in nmd mice that can partially suppress the phenotype, resulting in some animals that have extended life spans (up to 200 days). To eliminate the intrinsic complications of the C57BL/6 background and the variation in survival due to the genetic modifier effect, we created a new SMARD1 mouse model that contains the same intron 4 mutation in Ighmbp2 as nmd mice but is now on a FVB congenic background. FVB-nmd are consistently more severe than the original nmd mice with respect to survival, weigh and motor function. The relatively short life span (18-21 days) of FVB-nmd mice allows us to monitor therapeutic efficacy for a variety of novel therapeutics in a timely manner without the complication of the small percentage of longer-lived animals that were observed in our colony of nmd mice.

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. Here, we describe the generation and characterization of a new transgenic rat strain in which conditional expression of the ZsGreen fluorescent protein gene requires the presence of exogenous Cre recombinase. Breeding Cre-expressing rat strains to this stable ZsGreen reporter strain provides an ideal method for validating new rat Cre driver lines and will greatly accelerate the characterization pipeline.

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. Although these tools have been developed for over 30 years, they require additional improvement. Currently, there is no consensus on how to select the tools to answer the specific scientific questions of interest. Selecting the appropriate cell ablation technique to study the function of a targeted cell population is less straightforward than selecting the method to study a gene's functions. In this review, we discuss the features of the various tools for targeted cell ablation and provide recommendations for optimal application of specific approaches.