Single-step conversion of P1 and P1 artificial chromosome clones into yeast artificial chromosomes.
Large insert genomic clones are useful for generating transgenic animals, particularly when specific mutations are introduced. To facilitate manipulation of large genomic sequences, we developed a method of converting Escherichia coli P1 artificial chromosomes (PACs) into yeast artificial chromosomes (YACs). A shuttle vector, pMAX-121, was generated that contains elements needed to generate a YAC (cen4, ars, ura3, his, and two telomere segments) along with approximately 1.3 kb of sequence homologous to P1 and PAC vector sequences. Cotransformation of yeast with the target PAC or P1 clone and pMAX-121 results in two homologous recombination events. The first, between the target clone and pMAX-121, results in a circular molecule. The second is an intramolecular recombination event between the two pMAX-121 telomere sequences, resulting in a linear molecule. The resulting YAC is stably maintained in yeast and can be further modified using homologous recombination. The method was used to convert a 201-kb PAC containing the human tau gene into a stable linear YAC. A second vector, pLys2-neo, was developed to retrofit the YAC with the yeast lys2 gene, a selectable marker replacing the yeast ura3 gene, and a Pgk-neo cassette that confers G418 resistance to mammalian cells. The resulting YAC can be used for generating transgenic animals and stably transfected cell lines. Also, the lys2 marker facilitates introduction of mutations by homologous recombination.
Poorkaj
P
P
Geriatric Research Education Clinical Center, Seattle, Washington 98108, USA. pips@u.washington.edu
Peterson
K R
KR
Schellenberg
G D
GD
D017101:Bacteriophage P1 / Q000235:genetics*; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004274:DNA, Recombinant; D005822:Genetic Vectors / Q000235:genetics; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014170:Transformation, Genetic
D004274:DNA, Recombinant