Generation and analysis of partially haploid cells with Cre-mediated chromosome deletion in the lymphoid system

Abstract

The fast accumulation of mutant mouse strains in recent years has provided an invaluable resource for phenotype-based genetic screens. However, study of lymphoid phenotypes can be obscured or impractical if homozygous mutations cause early embryonic defects. To aid phenotype screening of germ line mutations in the lymphoid system, we developed a method to induce loss of heterozygosity (LOH) in developing lymphocytes through chromosome deletion. Chromosome deletion was triggered by Cre/loxP-mediated inverse sister chromatid recombination in the G(2)/M phase of the cell cycle, leading to the generation of daughter cells missing part of or the entire recombinant chromosome. We show that the resulting cells were viable and capable of additional rounds of cell division, thus providing raw materials for subsequent phenotypic assessment. We used the recombination system to induce LOH at the E2A locus in developing B cells. A significant loss of pro-B and pre-B cells was observed when the wild-type allele was removed by chromosome deletion from the E2A heterozygous mice, a result consistent with the required role for E2A in B cell development. We also demonstrated the effectiveness of Cre-mediated chromosome deletion in the LOH assay for HEB function in T cell development. Thus, the Cre-mediated chromosome deletion provides a new and effective method for genome-wide assessment of germ line mutations in the lymphoid system.

FIGURE 1.

a, Cre/lox-mediated sister chromatid recombination. Gray and black bars represent a pair of homologous chromosomes with the round ends as centromeres. A pair of inverted loxP sites (black triangles) separates the chromosome into proximal and distal parts. Cre-mediated recombination between the duplicated sister chromatids (gray) during G₂ phase could result in either inverted or reciprocal recombination. b, diagram of the recombination cassette in the MigR1CDV1 retroviral vector (where CD is chromosome deletion). The MigR1 retroviral vector was modified by inserting the puromycin resistance gene (Puro) between the 5′-long terminal repeat (5′ LTR) and the internal ribosome entry site-EGFP (Ires-gfp). The iloxP site was inserted between the puromycin and internal ribosome entry site-EGFP sequences. c, Cre-dependent loss of EGFP expression in a representative clone from MigR1CDV1-infected NIH-3T3 cell lines. Retrovirus carrying Cre-internal ribosome entry site-hCD2 was used in the second round of infection of MigR1CDV1 stable cell lines. hCD2⁻ (left panels) and hCD2⁺ (right panels) cells were sorted into separate culture wells at day 0 with a purity exceeding 95%. EGFP expression was checked from day 1 to 4 and is displayed in two-dimensional plots for hCD2 and EGFP expression. d, cell counts of the same culture in c from day 5 to 9 at 2-day intervals. Bar groups from left to right represent EGFP⁺/hCD2⁺, EGFP⁻/hCD2⁺, and EGFP⁺/hCD2⁻ subsets from sorted hCD2⁺ cultures.

FIGURE 2.

a, diagram of the iloxP recombination cassette on chromosome 9 in pbch9 transgenic mouse. The recombination cassette containing EGFP, iloxP, and hCD2 was integrated into chromosome 9 via piggyBac transposition. EGFP is located on the proximal side of chromosome 9, and hCD2 is on the distal side. Arrows indicate the direction of transcription. b, effect of LckCre on hCD2 expression from the pbch9 chromosome. The pbch9 transgenic line was crossed onto the LckCre background. Splenic T and B cells from representative mice of the indicated genotypes were gated with the T cell receptor β and B220 markers, respectively, prior to histogram analysis for hCD2 expression (n = 8). c, effect of mb1Cre on hCD2 expression. The experiment was performed as described for b (n = 4). d, karyotyping analysis of mitotic events in ex vivo bone marrow culture. Mitotic spreads representing the euploid state, chromosome 9 (Chr.9) breaks, and chromosomal 9 distal deletions are shown. Four pbch9;mb1Cre animals were analyzed by scoring 20–22 mitotic nuclei for each sample. All chromosomal abnormalities identified involve chromosome 9, which accounts for 15 ± 4% of the total score. PE-A, phycoerythrin area.

FIGURE 2.

a, diagram of the iloxP recombination cassette on chromosome 9 in pbch9 transgenic mouse. The recombination cassette containing EGFP, iloxP, and hCD2 was integrated into chromosome 9 via piggyBac transposition. EGFP is located on the proximal side of chromosome 9, and hCD2 is on the distal side. Arrows indicate the direction of transcription. b, effect of LckCre on hCD2 expression from the pbch9 chromosome. The pbch9 transgenic line was crossed onto the LckCre background. Splenic T and B cells from representative mice of the indicated genotypes were gated with the T cell receptor β and B220 markers, respectively, prior to histogram analysis for hCD2 expression (n = 8). c, effect of mb1Cre on hCD2 expression. The experiment was performed as described for b (n = 4). d, karyotyping analysis of mitotic events in ex vivo bone marrow culture. Mitotic spreads representing the euploid state, chromosome 9 (Chr.9) breaks, and chromosomal 9 distal deletions are shown. Four pbch9;mb1Cre animals were analyzed by scoring 20–22 mitotic nuclei for each sample. All chromosomal abnormalities identified involve chromosome 9, which accounts for 15 ± 4% of the total score. PE-A, phycoerythrin area.

FIGURE 3.

a, sorting criteria for bone marrow B cells used for CFSE staining. Cells were pre-gated on 7AAD⁻ and B220⁺ populations. hCD2⁺ cells were sorted from a Cre⁻ mouse, and both hCD2⁺ and hCD2⁻ cells were sorted from the same Cre⁺ mouse. b, CFSE analysis of cell proliferation. Cells were pre-gated on 7AAD⁻B220⁺CD19⁺ populations and checked for CFSE dilution and hCD2 expression after 3 days in culture. Data are representative of two independent experiments. FITC-A, fluorescein isothiocyanate area; IL-7, interleukin-7; 7AAD, 7-aminoactinomycin D; SSC, side scatter; APC, allophycocyanin; PE-A, phycoerythrin area.

FIGURE 4.

a, diagram of the pbch10 allele. The EGFP-iloxP-hCD2 cassette was integrated at ∼3 megabases upstream of the E2A gene on chromosome 10 (CH.10). hCD2 and E2A are both on the distal side of the iloxP site. b, hCD2 expression in B cells from different stages of development in the bone marrow and spleen of the indicated genotypes. A sequential gating of bone marrow cells from pbch10 mice separated the B cell population into prepro-B (B220⁺CD43⁺CD19⁻), pro-B (B220⁺CD43⁺CD19⁺), and a combination of pre-B and B (B220⁺CD43⁻) cells. B cells are defined as B220⁺IgM⁺ cells isolated from the spleen. These gating criteria were used to analyze hCD2 expression in the three genotype groups shown on the right. Histograms of hCD2 expression in B cell subsets are shown, with the vertical line indicating gating criteria to separate hCD2⁺ and hCD2⁻ cells. The relative percentage of the hCD2⁻ fraction is indicated in the plot. SA APC-Cγ7-A, streptavidin-allophycocyanin-cyanine 7 area; PE-A, phycoerythrin area. c, total cell counts of B cell subsets in the bone marrow of pbch10;mb1Cre mice (fine dot bars, n = 6) and pbch10/E2A^flox;mb1Cre mice (coarse dot bars, n = 8). d, cell counts of the hCD2⁻ population in the corresponding groups in c. e, total cell counts and hCD2⁻ cell counts of splenic B cells using the same mice analyzed in b–d. f, mean fluorescent intensity (MFI) of hCD2⁺ fractions in pro-B and pre-B/B cells of the indicated genotypes. **, p < 0.01; ***, p < 0.001.

FIGURE 5.

LOH test of the Heb locus with the pbch9 chromosome. The bar graphs summarize the percentage (upper graph) and absolute numbers (lower graph) of DN and DP thymocytes in the thymus. Total thymocytes were analyzed by co-staining with the CD4 and CD8 markers, which separate cells into DN, DP, and CD4 or CD8 single-positive fractions. Three groups of mice 1–2 months old were included in this analysis. cre⁻, LckCre transgene-negative controls (n = 5); heb^flox/+;cre⁺, Heb^flox/+;E2A^flox/flox;LckCre mice (n = 4); pbch9/heb^flox;cre⁺, pbch9/Heb^flox;E2A^flox/flox;LckCre mice (n = 5). *, p < 0.05; ***, p < 0.001.