Generation of mice with a conditional allele for Ift172

Abstract

Ift172 encodes a gene product that is part of a complex that mediates intraflagellar transport (IFT), a process necessary for the genesis and maintenance of cilia. Genetic studies in mice have offered evidence that Ift172 also plays a role in hedgehog signaling. Disruption of Ift172 in mice is associated with lethality at about embryonic day 11, limiting studies to understand the role for Ift172 in later development and the adult. To further our understanding of the later roles of Ift172, we have generated mice with a conditional allele for Ift172. We have confirmed the phenotype of the disrupted allele by using CRE expression directed by the prx1 enhancer to disrupt the conditional Ift172 allele in the developing limb. Keywords Intraflagellar transport; Hedgehog; Limb Development; CRE recombinaseThe intraflagellar transport protein, IFT172, is a member of a complex of proteins that are required for genesis and maintenance of primary cilia (Eggenschwiler and Anderson 2007). Disruption of intraflagellar transport by mutation of Ift genes is associated with several human pathologies including polycystic kidney disease (Yoder 2007). Mutation of the genes for the IFT proteins IFT88 or IFT172 also blocks hedgehog signaling (Huangfu et al. 2003). Subsequent studes have demonstrated that many hedgehog signaling components, including the Gli transcriptional regulators, must localize to the primary cilium for proper signaling to occur (Eggenschwiler and Anderson 2007). We originally cloned the cDNA encoding IFT172 in a yeast two-hybrid screen for proteins that interact with the LIM domains of the LIMhomeodomain transcription factor Lhx3 (Howard and Maurer 2000). Disruption of Ift172 causes lethality in mice at about embryonic day 11 (Huangfu et al. 2003;Gorivodsky et al. 2009). To further our understanding of the possible involvement of Ift172 in Lhx3-mediated pituitary signaling we have generated mice harboring a conditional allele for Ift172. This mouse model circumvents the embryonic lethality of the Ift172 null by allowing the tissue-specific or temporal disruption of Ift172. Materials and methods Construction of the Ift172 gene targeting vectorGenomic DNA containing the Ift172 gene was obtained by screening a λGT-10 library of 129/ SvJ gemonic DNA. A targeting vector in which exons 2 and 3 are flanked by loxP sites and which also contains neomycin and chloramphenicol resistance cassettes flanked by frt sites was prepared by homologous recombination in E. coli (Lee et al. 2001). Details of the construction of the targeting vector are available on request.The targeting vector was tested for proper design and function by subjecting it to FLP-mediated recombination in the bacterial stain EL250 and CRE mediated recombination in the bacterial strain EL350 (Lee et al. 2001). Generation of Mice Carrying the Conditional and Disrupted Ift172 AllelesThe completed targeting vector was linearized and electroporated into 129/SvJ embryonic stem cells and recombinant cells were selected with G418. Recombinant embryonic stem...

Function and regulation of primary cilia and intraflagellar transport proteins in the skeleton

“…In addition to the in vitro studies, some research groups have studied the in vivo role of cilia in the mesenchyme using Prx1‐Cre transgenic mice to delete the target gene in early limb bud mesenchyme and a subset of craniofacial mesenchyme . Interestingly, the deletion of Ift172 with Prx1‐Cre ( Ift172 f/t ;Prx1‐Cre, one allele is the germline deletion and the other is specifically deleted in the developing limb bud mesenchyme) causes eight digits on each forelimb and a single extra digit on each hindlimb . The deletion of Ift88 by using Prx1‐Cre ( Ift88 ;Prx1‐Cre) leads to polydactyly with the loss of anteroposterior digit patterning and shortening of the proximodistal axis .…”

Transcriptional profiling of C. elegans DAF-19 uncovers a ciliary base-associated protein and a CDK/CCRK/LF2p-related kinase required for intraflagellar transport

“…Examples of these disorders include the multi-systemic Bardet-Biedl and Meckel syndromes (Zaghloul and Katsanis, 2009), Polycystic Kidney Diseases (Haycraft et al, 2001), retinal degenerative diseases (Adams et al, 2007), and reproductive dysfunction (Rashid et al, 2010). In vertebrates, disruption of IFT proteins causes embryonic lethality and ciliopathy-associated proteins are often critical for early development, making it difficult to study some cilia-related proteins or processes (Cortellino et al, 2009; Howard et al, 2010). …”

Ofd1 Controls Dorso-Ventral Patterning and Axoneme Elongation during Embryonic Brain Development

“…Complete inactivation of Ift88 causes absence of primary cilia both during embryonic development and in adult life [85], [86], whereas the role of Ift172 in ciliogenesis in the adult life is still undefined [87]. Interestingly, recent data suggest that the assembly of primary cilia is a critical event in the dendritic refinement and synaptic integration of adult-born neurons [88].…”