Deciphering protein function during mitosis in PtK cells using RNAi

Jane R. Stout, Rania S. Rizk, Susan L. Kline, Claire Walczak

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

Background: Studying mitosis requires a system in which the dramatic movements of chromosomes and spindle microtubules can be visualized. PtK cells, due to their flat morphology and their small number of large chromosomes, allow microscopic visualizations to be readily performed. Results: By performing RNAi in PtK cells, we can explore the function of many proteins important for spindle assembly and chromosome segregation. Although it is difficult to transfect DNA into PtK cells (efficiency ∼ 10%), we have transfected a fluorescent siRNA at nearly 100% efficiency. Using a cDNA expression library, we then isolated a complete PtK MCAK (P-MCAK) cDNA. PMCAK shares 81% identity to Human-MCAK (H-MCAK) protein and 66% identity to H-MCAK DNA. Knockdown of P-MCAK by RNAi caused defects in chromosome congression and defective spindle organization. Live imaging revealed that chromosomes had defects in congression and segregation, similar to what we found after microinjection of inhibitory anti-MCAK antibodies. Because it is laborious to isolate full-length clones, we explored using RT-PCR with degenerate primers to yield cDNA fragments from PtK cells from which to design siRNAs. We isolated a cDNA fragment of the mitotic kinesin Eg5 from PtK cells. This fragment is 93% identical to H-Eg5 protein and 87% identical to H-Eg5 DNA. A conserved 21 bp siRNA was used for RNAi in both HeLa and PtK cells in which Eg5 knockdown resulted in an increased mitotic index and cells with monopolar spindles. In addition, we used RT-PCR to isolate fragments of 5 additional genes, whose sequence identity ranged from 76 to 90% with human, mouse, or rat genes, suggesting that this strategy is feasible to apply to any gene of interest. Conclusion: This approach will allow us to effectively probe mitotic defects from protein knockdowns by combining genomic information from other organisms with the tractable morphology of PtK cells.

Original languageEnglish
Article number26
JournalBMC cell biology [electronic resource]
Volume7
DOIs
StatePublished - Jun 23 2006

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RNA Interference
Mitosis
Chromosomes
Proteins
Complementary DNA
Small Interfering RNA
Genes
Kinesin
Polymerase Chain Reaction
Chromosome Segregation
Mitotic Index
DNA
Microinjections
Gene Library
HeLa Cells
Microtubules
Anti-Idiotypic Antibodies
Clone Cells

ASJC Scopus subject areas

  • Cell Biology

Cite this

Deciphering protein function during mitosis in PtK cells using RNAi. / Stout, Jane R.; Rizk, Rania S.; Kline, Susan L.; Walczak, Claire.

In: BMC cell biology [electronic resource], Vol. 7, 26, 23.06.2006.

Research output: Contribution to journalArticle

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