A simple method for obtaining cell-specific cDNA from small numbers of growing root-hair cells in Arabidopsis thaliana.
A simple and rapid method for cloning specific cDNAs from mRNA populations derived solely from small numbers of root-hair cells is described here. To identify genes expressed during the earliest visible stage of root-hair cell development, cell contents were aspirated from small numbers of Arabidopsis root-hair cells at or just before this stage. This material was used to make reusable solid-phase oligo-dT-primed cDNA libraries. To demonstrate that the libraries contained high quality longer cDNAs, a fragment located 2.7 kb from the 3' end of the cDNA of the single copy root-hair expressed gene RHD3 was cloned using a nested PCR strategy. This technique was also used to obtain novel gene expression information by cloning the full-length 0.85 kb cDNA of the Rop2 GTPase from this library. This approach offers a means of cloning larger cDNAs directly from small numbers of growing root-hair cells and, potentially, other epidermal cell types.
Jones
Mark A
MA
School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK. m.a.jones@ex.ac.uk
Grierson
Claire S
CS
D017360:Arabidopsis / Q000166:cytology / Q000235:genetics* / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics; D015723:Gene Library; D018517:Plant Roots / Q000166:cytology / Q000235:genetics* / Q000254:growth & development; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D020741:rho GTP-Binding Proteins / Q000235:genetics
D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D012333:RNA, Messenger; D019204:GTP-Binding Proteins; C464507:RHD3 protein, Arabidopsis; D020741:rho GTP-Binding Proteins