RNA Isolation from Xenopus Inner Ear Sensory Endorgans for Transcriptional Profiling and Molecular Cloning

Trujillo-Provencio, Casilda; Powers, TuShun R.; Sultemeier, David R.; Serrano, Elba E.

doi:10.1007/978-1-59745-523-7_1

Cited by 4 publications

(8 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Inner ear RNA was isolated from three groups of 5–10 juvenile X. laevis according to established methods [79]. We use the term “ replicate ” to refer to one of these samples of pooled inner ear RNA (10–19 inner ears each).…”

Section: Methodsmentioning

confidence: 99%

“…The SMART™ RACE cDNA Amplication Kit (Clontech) was used to confirm that genes detected on the microarray could be amplified with RT-PCR from juvenile X. laevis inner ear template RNA [79]. Primers for the coding regions of gata3, pfn2, six1, pmp 22 , clu, matn2, clcknb , and scnn1b were designed from Xl-PSID consensus sequences (see Additional file 12).…”

Section: Methodsmentioning

confidence: 99%

“…Negative controls for this experiment included both a “No RT” control (reactions with only template RNA and primers) and a “No cDNA” contamination control (reactions with primers and no RT product as template). Positive PCR products were purified with QIAquick PCR purification kit (Qiagen) and partial fragments were sequenced for gene verification on the ABI PRISM® 3100 Genetic Analyzer according to established procedures [79]. Sequence data were submitted to the NCBI GenBank archive [GenBank: JX033705, JX033706, JX033707, JX033708, JX033709, JX033710 , JX033711, JX035911].…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Probing the Xenopus laevis inner ear transcriptome for biological function

et al. 2012

Self Cite

View full text Add to dashboard Cite

BackgroundThe senses of hearing and balance depend upon mechanoreception, a process that originates in the inner ear and shares features across species. Amphibians have been widely used for physiological studies of mechanotransduction by sensory hair cells. In contrast, much less is known of the genetic basis of auditory and vestibular function in this class of animals. Among amphibians, the genus Xenopus is a well-characterized genetic and developmental model that offers unique opportunities for inner ear research because of the amphibian capacity for tissue and organ regeneration. For these reasons, we implemented a functional genomics approach as a means to undertake a large-scale analysis of the Xenopus laevis inner ear transcriptome through microarray analysis.ResultsMicroarray analysis uncovered genes within the X. laevis inner ear transcriptome associated with inner ear function and impairment in other organisms, thereby supporting the inclusion of Xenopus in cross-species genetic studies of the inner ear. The use of gene categories (inner ear tissue; deafness; ion channels; ion transporters; transcription factors) facilitated the assignment of functional significance to probe set identifiers. We enhanced the biological relevance of our microarray data by using a variety of curation approaches to increase the annotation of the Affymetrix GeneChip® Xenopus laevis Genome array. In addition, annotation analysis revealed the prevalence of inner ear transcripts represented by probe set identifiers that lack functional characterization.ConclusionsWe identified an abundance of targets for genetic analysis of auditory and vestibular function. The orthologues to human genes with known inner ear function and the highly expressed transcripts that lack annotation are particularly interesting candidates for future analyses. We used informatics approaches to impart biologically relevant information to the Xenopus inner ear transcriptome, thereby addressing the impediment imposed by insufficient gene annotation. These findings heighten the relevance of Xenopus as a model organism for genetic investigations of inner ear organogenesis, morphogenesis, and regeneration.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Probing the Xenopus laevis inner ear transcriptome for biological function

et al. 2012

Self Cite

View full text Add to dashboard Cite

show abstract

“…Dissection methods for isolation of inner ear tissue from juvenile X. laevis and the method of total RNA isolation were according to the procedures outlined in Trujillo-Provencio et al (2009). The methods performed with X. laevis were in compliance with the Institutional Animal Care and Use Committee of New Mexico State University.…”

Section: Methodsmentioning

confidence: 99%

“…We are interested in probing the function of genes expressed in the Xenopus laevis inner ear that have been identified through cDNA library construction, RACE, and microarray analysis (Varela-Ramírez et al 1998; Serrano et al 2001; Powers et al 2007; Sultemeier et al 2005). Alternative systems for genetic analysis can advance such studies because of the relatively small size and extreme inaccessibility of inner ear organs (Díaz et al 1995; Trujillo-Provencio et al 2009). Our aim is to characterize inner ear genes through heterologous expression in cell lines.…”

Section: Introductionmentioning

confidence: 99%

Optimization of gene delivery methods in Xenopus laevis kidney (A6) and Chinese hamster ovary (CHO) cell lines for heterologous expression of Xenopus inner ear genes

Ramirez-Gordillo

Trujillo-Provencio

Knight

et al. 2011

In Vitro Cell.Dev.Biol.-Animal

Self Cite

View full text Add to dashboard Cite

The Xenopus inner ear provides a useful model for studies of hearing and balance because it shares features with the mammalian inner ear, and because amphibians are capable of regenerating damaged mechanosensory hair cells. The structure and function of many proteins necessary for inner ear function have yet to be elucidated and require methods for analysis. To this end, we seek to characterize Xenopus inner ear genes outside of the animal model through heterologous expression in cell lines. As part of this effort, we aimed to optimize physical (electroporation), chemical (lipid-mediated; Lipofectamine™ 2000, Metafectene® Pro), and biological (viral-mediated; BacMam virus Cellular Lights™ Tubulin-RFP) gene delivery methods in amphibian (Xenopus; A6) cells and mammalian (Chinese hamster ovary (CHO)) cells. We successfully introduced the commercially available pEGFP-N3, pmCherry-N1, pEYFP-Tubulin, and Cellular Lights™ Tubulin-RFP fluorescent constructs to cells and evaluated their transfection or transduction efficiencies using the three gene delivery methods. In addition, we analyzed the transfection efficiency of a novel construct synthesized in our laboratory by cloning the Xenopus inner ear calcium-activated potassium channel β1 subunit, then subcloning the subunit into the pmCherry-N1 vector. Every gene delivery method was significantly more effective in CHO cells. Although results for the A6 cell line were not statistically significant, both cell lines illustrate a trend towards more efficient gene delivery using viral-mediated methods; however the cost of viral transduction is also much higher. Our findings demonstrate the need to improve gene delivery methods for amphibian cells and underscore the necessity for a greater understanding of amphibian cell biology.

show abstract

Transcriptomic Analysis Identifies Candidate Genes for Differential Expression duringXenopus laevisInner Ear Development

Virk,

Trujillo-Provencio,

Serrano

2024

Preprint

Self Cite

View full text Add to dashboard Cite

Background.The genes involved in inner ear development and maintenance of the adult organ have yet to be fully characterized. Previous genetic analysis has emphasized the early development that gives rise to the otic vesicle. This study aimed to bridge the knowledge gap and identify candidate genes that are expressed as the auditory and vestibular sensory organs continue to grow and develop until the systems reach postmetamorphic maturity.Methods.Affymetrix microarrays were used to assess inner ear transcriptome profiles from threeXenopus laevisdevelopmental ages where all eight endorgans comprise mechanosensory hair cells: larval stages 50 and 56, and the post-metamorphic juvenile. Pairwise comparisons were made between the three developmental stages and the resulting differentially expressedX. laevisProbe Set IDs (Xl-PSIDs) were assigned to four groups based on differential expression patterns. DAVID analysis was undertaken to impart functional annotation to the differentially regulated Xl-PSIDs.Results.Analysis identified 1510 candidate genes for differential gene expression in one or more pairwise comparison. Annotated genes not previously associated with inner ear development emerged from this analysis, as well as annotated genes with established inner ear function, such as oncomodulin, neurod1, and sp7. Notably, 36% of differentially expressed Xl-PSIDs were unannotated.Conclusions.Results draw attention to the complex gene regulatory patterns that characterizeXenopusinner ear development, and underscore the need for improved annotation of theX. laevisgenome. Outcomes can be utilized to select candidate inner ear genes for functional analysis, and to promoteXenopusas a model organism for biomedical studies of hearing and balance.

show abstract

RNA Isolation from Xenopus Inner Ear Sensory Endorgans for Transcriptional Profiling and Molecular Cloning

Cited by 4 publications

References 8 publications

Probing the Xenopus laevis inner ear transcriptome for biological function

Probing the Xenopus laevis inner ear transcriptome for biological function

Optimization of gene delivery methods in Xenopus laevis kidney (A6) and Chinese hamster ovary (CHO) cell lines for heterologous expression of Xenopus inner ear genes

Transcriptomic Analysis Identifies Candidate Genes for Differential Expression duringXenopus laevisInner Ear Development

Contact Info

Product

Resources

About