Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning

Abstract

The transformation-associated recombination (TAR) cloning technique allows selective and accurate isolation of chromosomal regions and genes from complex genomes. The technique is based on in vivo recombination between genomic DNA and a linearized vector containing homologous sequences, or hooks, to the gene of interest. The recombination occurs during transformation of yeast spheroplasts that results in the generation of a yeast artificial chromosome (YAC) containing the gene of interest. To further enhance and refine the TAR cloning technology, we determined the minimal size of a specific hook required for gene isolation utilizing the Tg.AC mouse transgene as a targeted region. For this purpose a set of vectors containing a B1 repeat hook and a Tg.AC-specific hook of variable sizes (from 20 to 800 bp) was constructed and checked for efficiency of transgene isolation by a radial TAR cloning. When vectors with a specific hook that was >/=60 bp were utilized, approximately 2% of transformants contained circular YACs with the Tg.AC transgene sequences. Efficiency of cloning dramatically decreased when the TAR vector contained a hook of 40 bp or less. Thus, the minimal length of a unique sequence required for gene isolation by TAR is approximately 60 bp. No transgene-positive YAC clones were detected when an ARS element was incorporated into a vector, demonstrating that the absence of a yeast origin of replication in a vector is a prerequisite for efficient gene isolation by TAR cloning.

Figure 1

Construction of TAR cloning vectors containing a different length of homology to the Tg.AC transgene. (A) The structure of the Tg.AC transgene. The transgene unit consists of a ζ-globin promoter fused to the v-Ha-ras structural gene with a terminal SV40 polyadenylation signal sequence (5,20). Nine different sizes of targeting sequence (from 800 to 20 bp), shown by filled bars, were selected from the 3′-terminal region of the transgene containing an SV40 polyadenylation signal sequence. (B) A scheme of a TAR vector. A set of TAR vectors with targeting sequences of different sizes (800, 600, 373, 284, 186, 118, 60, 40 and 20 bp) homologous to the 3′-end of the transgene was constructed. Each vector contains a 130 bp B1 repeat as a second targeting sequence. Vectors were linearized by SalI restriction endonuclease before transformation.

Figure 2

A scheme of isolation of the Tg.AC transgene as a series of circular YACs using a TAR cloning vector containing a 3′ Tg.AC sequence and a B1 repeat. Yeast spheroplasts were transformed with genomic mouse DNA along with a TAR cloning vector containing a 3′ sequence (dotted box) and a B1 at the ends of the linearized plasmid. Recombination between sequences in the vector and genomic DNA containing the transgene leads to the establishment of circular YACs that extend from the 3′ sequence to various B1 positions. Because the vector lacks ARS, the only YACs that will be stably maintained are those that included mouse DNA fragments containing a yeast ARS-like sequence. CEN6 corresponds to the yeast chromosome VI centromere and HIS3 is a selectable marker. Arrows indicate positions of primers (specific for a ζ-globin promoter region) used for detection of positive clones among primary transformants.