Ex Vivo Magnetofection: A Novel Strategy for the Study of Gene Function in Mouse Organogenesis

Terje Svingen, Dagmar Wilhelm, Alexander N. Combes, Brett Hosking, Vincent R. Harley, Andrew H. Sinclair, Peter Koopman

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Gene function during mouse development is often studied through the production and analysis of transgenic and knockout models. However, these techniques are time- and resource-consuming, and require specialized equipment and expertise. We have established a new protocol for functional studies that combines organ culture of explanted fetal tissues with microinjection and magnetically induced transfection ("magnetofection") of gene expression constructs. As proof-of-principle, we magnetofected cDNA constructs into genital ridge tissue as a means of gain-of-function analysis, and shRNA constructs for loss-of-function analysis. Ectopic expression of Sry induced female-to-male sex-reversal, whereas knockdown of Sox9 expression caused male-to-female sex-reversal, consistent with the known functions of these genes. Furthermore, ectopic expression of Tmem184a, a gene of unknown function, in female genital ridges, resulted in failure of gonocytes to enter meiosis. This technique will likely be applicable to the study of gene function in a broader range of developing organs and tissues. Developmental Dynamics 238:956-964, 2009. (C) 2009 Wiley-Liss, Inc.
Original languageEnglish
JournalDevelopmental Dynamics
Volume238
Issue number4
Pages (from-to)956-964
Number of pages9
ISSN1058-8388
DOIs
Publication statusPublished - 2009
Externally publishedYes

Keywords

  • Animals
  • Cell Line
  • Embryo, Mammalian
  • Female
  • Gene Expression Regulation, Developmental
  • Humans
  • Male
  • Mice
  • Organogenesis
  • Ovary
  • RNA Interference
  • SOX9 Transcription Factor
  • Sex Chromosomes
  • Sex-Determining Region Y Protein
  • Testis
  • Tissue Culture Techniques
  • Sox9 protein, mouse
  • Sry protein, mouse
  • complementary DNA
  • membrane protein
  • plasmid DNA
  • short hairpin RNA
  • testis determining factor
  • transcription factor Sox9
  • transmembrane protein 184a
  • unclassified drug
  • animal cell
  • animal experiment
  • article
  • cell fate
  • embryo
  • ex vivo study
  • explant
  • female
  • gene expression
  • gene function
  • genetic transfection
  • germ cell
  • human
  • human cell
  • karyotype 46,XX
  • karyotype 46,XY
  • magnetofection
  • male
  • meiosis
  • mouse
  • nonhuman
  • organ culture technique
  • organogenesis
  • ovary
  • ovary development
  • priority journal
  • protein expression
  • sex transformation
  • testis development
  • Gene delivery
  • Germ cells
  • Meiosis
  • Sex determination
  • Sox9
  • Sry
  • Tmem184a
  • gene delivery
  • sex determination
  • germ cells
  • ANATOMY
  • DEVELOPMENTAL
  • SEX DETERMINATION REVEALS
  • PRIMORDIAL GERM-CELLS
  • SRY-RELATED GENE
  • IN-VIVO
  • TESTIS DEVELOPMENT
  • DIFFERENTIAL EXPRESSION
  • CAMPOMELIC DYSPLASIA
  • TRANSCRIPTION FACTOR
  • OVARIAN DEVELOPMENT
  • CHICKEN EMBRYOS
  • Rodentia Mammalia Vertebrata Chordata Animalia (Animals, Chordates, Mammals, Nonhuman Vertebrates, Nonhuman Mammals, Rodents, Vertebrates) - Muridae [86375] mouse common female, male
  • mouse Sox9 gene [Muridae] mutation, expression
  • mouse Sry gene [Muridae] expression
  • mouse Tmem184a gene [Muridae] expression
  • cDNA constructs
  • gene expression constructs
  • short hairpin RNA constructs shRNA constructs
  • 02506, Cytology - Animal
  • 03502, Genetics - General
  • 03506, Genetics - Animal
  • 16504, Reproductive system - Physiology and biochemistry
  • 25502, Development and Embryology - General and descriptive
  • 32500, Tissue culture, apparatus, methods and media
  • Development
  • Methods and Techniques
  • Molecular Genetics
  • Reproductive System
  • gene function study
  • organism development
  • sex-reversal
  • Biochemistry and Molecular Biophysics
  • Reproduction
  • genital ridge tissue reproductive system
  • gonocyte reproductive system
  • ex vivo magnetofection laboratory techniques, genetic techniques
  • gain-of-function analysis laboratory techniques, genetic techniques
  • gene function analysis laboratory techniques, genetic techniques
  • loss-of-function analysis laboratory techniques, genetic techniques
  • magnetically induced transfection laboratory techniques, genetic techniques
  • microinjection laboratory techniques, genetic techniques
  • organ culture laboratory techniques, culturing techniques

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