Download Construction and stable transformation of Tetrahymena

Construction and stable transformation of Tetrahymena macronuclear artificial
chromosome (TtAC) for a large gene/DNA cloning and expression system
Ayça Fulya Üstüntanır and Muhittin Arslanyolu
Graduate School of Sciences, Biology Program, Anadolu University, Eskisehir, Turkey
Department of Biology, Molecular Biotechnology and Enzymology Laboratory, Faculty of
Sciences, Anadolu University, Eskisehir, Turkey.
In 1983, first artificial chromosome vector was constucted by using Tetrahymena telomere,
yeast centromere and replication origin to mimick eukaryotic chromosomes of Saccharomyces
cerevisiae. Artificial chromosomes (AC) with a capacity to carry 50-500 kb large DNA
fragment have been used in a construction of genomic library, chromosome walking and
functional complementation. The model organism T. thermophila has 24.725 genes, 12.000 of
them has no known biological function including some human homologs. In the future, one of
main goals of Tetrahymena laboratories will be characterizing the unknown biological function
of these genes in lifecycle by using functional complementation analysis based on phenotype
with transfering genomic DNA library into mutant Tetrahymena cells using AC based vectors.
There is no published literature describing an artificial chromosome for T.thermophila,
Therefore, the aim of this study is to construct T.thermophila Macronuclear Artificial
Chromosome (TtAC) which have telomere, rRNA replication origin and codon optimized neo4
cassette. This vector will be used as cloning vector for E.coli in the circular form and then turn
into linear expression vector in Tetrahymena thermophila mutants. In this study, C3-type origin
were amplified by PCR from pH4T2 vector and ligated to pNeo4 and pNeo3 vectors. Then,
Tetrahymena telomere cassette were amplified from pPXV-GFP vector of Paramecium and
were ligated to the constructed pNeo4-ori vector. These vectors (pNeo4-ori, pNeo3ori, circular
pNeo4oritel and linear pNeo4oritel) were transformed with biolistic gun-mediated
transformation into vegetative Tetrahymena (CU428) and then were also transformed into
conjugative Tetrahymena (CU428xB2086) with electoroporation. After the dilution and
distribution of cells with 1 μg/ml CdCl2 and 100 µg/ml Paromomycin into 96-well plate,
transforments were recorded daily up to 7 day for true selection with 1 μg/ml CdCl2, 600 µg/ml
Paromomycin. Total tranformant cell number were estimated based upon the Poisson
distrubition. Vector copy number were analysed with Real-time PCR. As a result, while
electrotransformation efficiency of linear pNeo4oritel artificial chromosome is 522
transformants/per µg DNA, biolistic gun transformation efficiency is 1016 transformants/µg
DNA. Analysis of real-time PCR data has shown that all vector constructs in cell slowly
disappeared in two months although they are under paromomycine selection. Based on these
results, we start to reconstruct a much better design of Tetrahymena artificial chromosome
(TtAC) mimicking the native structure by using alternative replication origine and orientation
of the elements.
Keywords: Tetrahymena thermophila, Artificial Chromosome, Telomere, Neomycin