MICER and Insertion Vectors

One important practical limitation for chromosome engineering is construction of targeting vectors to introduce the recombination cassettes to the rearrangement endpoints. Previously, genomic DNA libraries in ^-phage were screened with specific probes in order to isolate genomic DNA fragments to serve as the homology regions in gene targeting. Targeting vectors are then constructed by ligating two homology arms to one of the recombination substrates (5' or 3' HPRT-loxP cassettes). However, it could take one person a couple of months to build a targeting vector with this approach. To improve the efficiency of this step, the Bradley laboratory at Baylor College of Medicine constructed two mouse genomic phage libraries in which 10-kb genomic DNA fragments are cloned into two complementary backbones (Fig. 2)

(39). The cloning backbone for the first library has the 5' HPRT-loxP cassette, a PGKNeobpA positive selection cassette, and the Tyrosinase coat color gene for easy identification of the mice (Fig. 2A). On the other hand, the 3' HPRT-loxP cassette, the PGKpurobpA selection cassette, and the Agouti coat color gene are cloned into the backbone of the 3' HPRT library (Fig. 2B) (39). As a result, clones from these two libraries can be used directly as premade gene targeting vectors for chromosome engineering. Recently, The Wellcome Trust Sanger Institute has sequenced the two HPRT-loxP genomic libraries (Mutagenic Insertion and Chromosome Engineering Resource [MICER]) (http://www.sanger.ac.uk/PostGenomics/mousegenomics/)

(40). Individual MICER clones can now be viewed directly on the Ensembl website and ordered through the Sanger Institute (http://www.sanger.ac.uk/cgi-bin/teams/team38/ CloneRequest/CloneRequest).

To make a defined chromosome rearrangement, MICER clones corresponding to the two endpoints are linearized within the genomic inserts to generate a DNA double strand-break that stimulates efficient homologous recombination (Fig. 2). Gene targeting in ES cells with these vectors has been shown to be very efficient (40). Targeting events in ES cells using MICER clones can be identified using a DNA fragment that is removed from the genomic DNA insert in the linearization process, or using an external probe, in Southern hybridization (39,41).

Although MICER clones provide a convenient way to make targeting vectors for chromosome engineering, they are all insertion targeting vectors that can introduce challenges compared to replacement vectors (42). Because there is no negative selection marker present on insertion vectors, on homologous recombination, these vectors can potentially form concatemers that may greatly complicate analysis of targeted events. Concatemerization of the recombination substrates may also complicate the ensuing Cre-loxP recombination because multiple loxP sites in a concatemer might have different orientations.

To obtain clean targeting events, replacement vectors are usually used. Genomic DNA fragments serving as the two homology-arms are isolated so that the recombination cassettes can be placed between them. This can be difficult in many cases because appropriate restriction enzyme sites have to be found or engineered in order to ligate all the DNA fragments together.

Fig. 2. Mutagenic Insertion and Chromosome Engineering Resource (MICER) clones for chromosome engineering. (A) Clones from the first MICER library carry the 5'HPRTcassette, Neo and a Tyrosinase minigene (Ty). (B) Clones from the second MICER library have the 3' HPRT cassette, Puromycin resistance cassette (Puro) and a K14-agouti (Ag) cassette. Vectors are linearized in the middle of the genomic fragments (a, b, c, d) to create a DNA double strand break for homologous recombination. Filled arrow, loxP site.

Fig. 2. Mutagenic Insertion and Chromosome Engineering Resource (MICER) clones for chromosome engineering. (A) Clones from the first MICER library carry the 5'HPRTcassette, Neo and a Tyrosinase minigene (Ty). (B) Clones from the second MICER library have the 3' HPRT cassette, Puromycin resistance cassette (Puro) and a K14-agouti (Ag) cassette. Vectors are linearized in the middle of the genomic fragments (a, b, c, d) to create a DNA double strand break for homologous recombination. Filled arrow, loxP site.

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