Rapid generation of nested chromosomal deletions on mouse chromosome 2

Abstract

Nested chromosomal deletions are powerful genetic tools. They are particularly suited for identifying essential genes in development either directly or by screening induced mutations against a deletion. To apply this approach to the functional analysis of mouse chromosome 2, a strategy for the rapid generation of nested deletions with Cre recombinase was developed and tested. A loxP site was targeted to the Notch1 gene on chromosome 2. A targeted line was cotransfected with a second loxP site and a plasmid for transient expression of Cre. Independent random integrations of the second loxP site onto the targeted chromosome in direct repeat orientation created multiple nested deletions. By virtue of targeting in an F(1) hybrid embryonic stem cell line, F(1)(129S1xCast/Ei), the deletions could be verified and rapidly mapped. Ten deletions fell into seven size classes, with the largest extending six or seven centiMorgans. The cytology of the deletion chromosomes were determined by fluorescent in situ hybridization. Eight deletions were cytologically normal, but the two largest deletions had additional rearrangements. Three deletions, including the largest unrearranged deletion, have been transmitted through the germ line. Several endpoints also have been cloned by plasmid rescue. These experiments illustrate the means to rapidly create and map deletions anywhere in the mouse genome. They also demonstrate an improved method for generating nested deletions in embryonic stem cells.

Figure 1

Strategy for generating deletions. (A) To catalyze a deletion Cre recombinase requires a pair of loxP sites (arrows) on the same chromosome in the same orientation. One loxP vector, called the anchor, is inserted by targeting. The other loxP vector, called the secondary vector, is inserted at random. Cre recombinase (shown separately here but in practice introduced into cells at the same time as the secondary insertions) catalyzes the deletions. (B) The targeted anchor vector contains a neo-loxP-tk cassette. The secondary insertion vector, containing a tk-loxP-puro cassette, then is inserted at random. In the configuration illustrated, the two tk genes are internal to the deleted segment and removed: deletions therefore survive negative selection against the two tk genes. The neo and puro genes are included to select for the targeted and the random insertions, respectively.

Figure 2

Strategy for generation of nested deletions on mouse chromosome 2 by a combined targeted/random approach. (A) The Notch1 gene was targeted to insert a cassette consisting of neo, a loxP site, and tk. A targeted line was selected, and a linearized vector consisting of a stuffer fragment of bacterial DNA, tk, a loxP site, puro, and Bluescript plasmid (tk-loxP-puro) was introduced by electroporation along with a plasmid to transiently express Cre (the Cre plasmid is not shown). In a small fraction of random integrations, tk-loxP-puro is expected to insert in the orientation shown, generating a chromosome that could delete the genes between the two loxP sites. Both tk genes are lost with the deletion, generating cells that are resistant to selection for the neo (G418^R) and puro (puromycin^R) genes and against the tk genes (FIAU^R). Cells that haven't integrated the tk-loxP-puro vector, or cells that have integrated the second vector but have not deleted the two tk genes, will not survive selection with all three drugs. Probes and predicted fragments for confirmation of targeting, deletion and plasmid rescue of the deletion junction are shown. (B) Verification of targeting, deletion, and junction fragment rescue by Southern blot. Analysis of a targeted line and a deleted line with a probe from the 5′ end of Notch1 shows that the 5′ end of the wild-type (16 kb) and targeted (11 kb) Notch1 alleles are present in both the targeted and deleted lines. However, the 3′ end of the targeted Notch1 allele is missing in the deleted line as shown by the absence of an 8-kb XbaI fragment. The tk gene is present in the targeted line (3-kb XbaI fragment), but is absent from the deleted line, as expected. A probe to puro detects a 5.5-kb fragment in the deleted line, and an XbaI fragment of the same size is detected with a probe cloned by plasmid rescue. The rescued fragment probe from deletion 5 detects a 1-kb XbaI fragment in the targeted line, and fragments of 1 kb and 5.5 kb of half intensity in the deleted line, consistent with the proposed deletion event. The neo and puro genes are present on the same 4-kb KpnI fragment in the deletion line and tk is absent in the deletion line, consistent with the predicted Cre-catalyzed recombination between the loxP sites of the two vectors. (K: KpnI; X: XbaI)

Figure 3

Ten chromosome 2 deletions of seven different size classes were recovered. Verification and mapping of the deletions was performed by analysis of marker loss in cell line DNA. Because the deletions were made in an ES cell line derived from an F₁ hybrid embryo (129S1 × Cast/Ei), deletions could be mapped with polymorphic markers. (A) The 129S1 allele of D2Wsu32e was lost (−) in six of the 10 lines, indicating deletions on the 129S1 chromosome 2 of these cell lines. (B) The results for markers in the vicinity of Notch1 in the 10 cell lines are summarized and the deletions are arranged from largest to smallest. Note that only 129S1 alleles were lost. The vertical lines in B separate bins of markers. Markers within bins are not ordered relative to each other within the bins.

Figure 4

Most deletion chromosomes do not have secondary rearrangements. Verification of cytology and mapping of deletions was performed by FISH with YAC probes to chromosome 2. Hybridization signals on chromosome 2 are indicated with arrows; signals without arrows are on chromosomes other than 2 and are caused by YAC chimerism. (A) Deletion line 3 has two chromosomes 2, both with signals for each of YACs 144F11 (green, 9 cM), 158D10 (red, 15 cM), and 188A3 (yellow, 98 cM). (B) Deletion line 3 has signal for YAC 188A3 (green, 98 cM) on both chromosomes 2, but is missing signal for 456A8 (red, 14 cM) on one. (C) Deletion line 3 again has two signals for 188A3 (green, 98 cM), but only one chromosome 2 signal for YAC 304E7 (red, 13 cM). (D) In deletion line 10, one chromosome 2 has an inverted duplication containing 406F11 (red, 38 cM) and 88C11 (green, 48 cM).

Figure 5

Summary of chromosome 2 deletions. The deletions are arrayed from largest to smallest, and the markers are shown at the top. Line 7 had the only discrepancies between the results obtained with polymorphic markers and the results obtained by FISH. Line 7 was deleted for marker D2Mit318 but retained the YAC that is associated with it, 456A8. Line 7 also was deleted for D2Mit32 and could not be unambiguously scored for its YAC, 304E7. All lines have 40 chromosomes.