Gene transfer into the chicken auditory organ by in ovo microelectroporation

Research output: Contribution to journalArticle

Abstract

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2´-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immunolabeling and click EdU chemistry is provided.

Original languageEnglish (US)
Article numbere53864
JournalJournal of Visualized Experiments
Volume2016
Issue number110
DOIs
StatePublished - Apr 1 2016

Fingerprint

Gene transfer
Electroporation
Cell proliferation
Chickens
Assays
Genes
Plasmids
Mammals
Embryonic Structures
Cell Proliferation
Inner Ear
RNA
Embryonic Development
DNA
Green Fluorescent Proteins
Cells
Thymidine
Proteins
Auditory Cortex
Gene Targeting

Keywords

  • Auditory organ
  • Basilar papilla
  • Chicken
  • Developmental biology
  • Developmental biology
  • Differentiation
  • Gain- and loss-of-function
  • Hair cells
  • In ovo electroporation
  • Inner ear
  • Issue 110
  • Neuroscience
  • Otic vesicle
  • Proliferation

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)

Cite this

Gene transfer into the chicken auditory organ by in ovo microelectroporation. / Evsen, Lale; Doetzlhofer, Angelika.

In: Journal of Visualized Experiments, Vol. 2016, No. 110, e53864, 01.04.2016.

Research output: Contribution to journalArticle

@article{9e35a47e02354dad9314197907cf70ad,
title = "Gene transfer into the chicken auditory organ by in ovo microelectroporation",
abstract = "Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2´-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immunolabeling and click EdU chemistry is provided.",
keywords = "Auditory organ, Basilar papilla, Chicken, Developmental biology, Developmental biology, Differentiation, Gain- and loss-of-function, Hair cells, In ovo electroporation, Inner ear, Issue 110, Neuroscience, Otic vesicle, Proliferation",
author = "Lale Evsen and Angelika Doetzlhofer",
year = "2016",
month = "4",
day = "1",
doi = "10.3791/53864",
language = "English (US)",
volume = "2016",
journal = "Journal of Visualized Experiments",
issn = "1940-087X",
publisher = "MYJoVE Corporation",
number = "110",

}

TY - JOUR

T1 - Gene transfer into the chicken auditory organ by in ovo microelectroporation

AU - Evsen, Lale

AU - Doetzlhofer, Angelika

PY - 2016/4/1

Y1 - 2016/4/1

N2 - Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2´-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immunolabeling and click EdU chemistry is provided.

AB - Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2´-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immunolabeling and click EdU chemistry is provided.

KW - Auditory organ

KW - Basilar papilla

KW - Chicken

KW - Developmental biology

KW - Developmental biology

KW - Differentiation

KW - Gain- and loss-of-function

KW - Hair cells

KW - In ovo electroporation

KW - Inner ear

KW - Issue 110

KW - Neuroscience

KW - Otic vesicle

KW - Proliferation

UR - http://www.scopus.com/inward/record.url?scp=84964865178&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84964865178&partnerID=8YFLogxK

U2 - 10.3791/53864

DO - 10.3791/53864

M3 - Article

C2 - 27167684

AN - SCOPUS:84964865178

VL - 2016

JO - Journal of Visualized Experiments

JF - Journal of Visualized Experiments

SN - 1940-087X

IS - 110

M1 - e53864

ER -