Humanized c-Myc mouse.

BACKGROUND: A given tumor is usually dependent on the oncogene that is activated in the respective tumor entity. This phenomenon called oncogene addiction provides the rationale for attempts to target oncogene products in a therapeutic manner, be it by small molecules, by small interfering RNAs (siRNA) or by antigen-specific T cells. As the proto-oncogene product is required also for the function of normal cells, this raises the question whether there is a therapeutic window between the adverse effects of specific inhibitors or T cells to normal tissue that may limit their application, and their beneficial tumor-specific therapeutic action. To address this crucial question, suitable mouse strains need to be developed, that enable expression of the human proto-oncogene not only in tumor but also in normal cells. The aim of this work is to provide such a mouse strain for the human proto-oncogene product c-MYC. PRINCIPAL FINDINGS: We generated C57BL/6-derived embryonic stem cells that are transgenic for a humanized c-Myc gene and established a mouse strain (hc-Myc) that expresses human c-MYC instead of the murine ortholog. These transgenic animals harbor the humanized c-Myc gene integrated into the endogenous murine c-Myc locus. Despite the lack of the endogenous murine c-Myc gene, homozygous mice show a normal phenotype indicating that human c-MYC can replace its murine ortholog. CONCLUSIONS: The newly established hc-Myc mouse strain provides a model system to study in detail the adverse effects of therapies that target the human c-MYC protein. To mimic the clinical situation, hc-Myc mice may be cross-bred to mice that develop tumors due to overexpression of human c-MYC. With these double transgenic mice it will be possible to study simultaneously the therapeutic efficiency and adverse side effects of MYC-specific therapies in the same mouse

Confirmation of homologous recombination and c-MYC2 expression in ES cell clones.

<p>(A) Genomic DNA of ES cell clones 1, 14, 18 and 19 and of wildtype ES cells (wt Bruce 4) was digested with <i>Eco</i>RI. Digested DNA was analyzed by Southern blotting with a 5′ probe and a 3′ probe. (wt) DNA fragment of the wildtype <i>c-Myc</i> locus; (rec.) DNA-fragment of recombined <i>hc-Myc</i> locus. (B) Protein extracts were prepared of ES cell clones 1, 14, 18 and 19 as well as of wildtype ES cells (wt Bruce 4) and of a human lymphoblastoid cell line (LCL 1.11). Human c-MYC2 (hu. c-MYC, ca. 62 kDa) was detected with antibody clone Y69. In wildtype ES cells murine c-MYC2 (mu. c-MYC, ca. 64 kDa) was detected. For loading control an antibody specific for glyceraldehyde-3-phosphat-dehydrogenase (GAPDH; ca. 36 kDa) was used. Western blot results were reproduced five times.</p

Mice transgenic for <i>hc-Myc</i>.

<p>(A) Example of a chimeric animal (chimerism of 60 to 70%) that was generated by injection of ES cell clone 14 into Balb/c blastocysts (day 3.5). (B) Chimeric animals were cross-bred to wildtype B6 mice yielding black progeny. Brother and sister matings of <i>hc-Myc</i> heterozygous mice (tested by PCR analysis) gave rise to <i>hc-Myc</i> homozygous offspring. The picture represents a PCR analysis of the genotypes of the offspring. For analysis genomic DNA was isolated from tail tissue. As controls genomic DNA of ES cell clones 1 and 14 was used. mu.c-Myc: PCR product of murine <i>c-Myc</i>. hc-Myc: PCR product of <i>hc-Myc</i>. (C) Detection of human c-MYC2 (hu. c-MYC, 62 kDa) in splenic cells of homozygous hc-Myc mice by Western blotting. In splenic cells of wildtype mice murine c-MYC2 was detected (mu. c-MYC, ca. 64 kDa). For positive control protein extracts from a human lymphoblastoid cell line (LCL 1.11) were used. Glyceraldehyde-3-phosphat-dehydrogenase (GAPDH, 36 kDa) was used as loading control.</p

No difference in the rate of proliferation of splenic hc-Myc and wildtype B cells.

<p>Splenic B cells were isolated by magnetic cell separation from homozygous hc-Myc mice (B6 hc-Myc^+/+) and wildtype B6 mice (B6 wt). For analyzing rate of proliferation, CFSE-stained splenic B cells were stimulated with LPS or anti-CD40 and IL-4. Proliferation was analyzed on day 1 and day 3 by flow cytometric analysis. Depicted are analyzes of living cells (TO-PRO-3 negative) on day 1 and day 3.</p

hc-Myc mice have normal body and organ weights.

<p>Body weight (A) and organ weight of spleen (B), both kidneys (C) and liver (D) were analyzed of heterozygous (B6 hc-Myc^+/−, n = 5) and homozygous hc-Myc (B6 hc-Myc^+/+, n = 8) mice as well as of wildtype B6 mice (B6 wt, n = 8) at the age of 10 - 11 weeks. Each symbol represents one mouse. The mean weight is indicated in the graphs with a black line and shown at the top of the symbols. Where ever small differences were observed, they were not significant.</p

Normal cell numbers in lymphoid organs of hc-Myc mice as compared to wildtype mice.

<p>Cell numbers in the spleen (A), both inguinal lymph nodes (B) and bone marrow of one tibia (C) was analyzed of 10 – 11 week old heterozygous (B6 hc-Myc^+/−, n = 5), homozygous (B6 hc-Myc^+/+, n = 5) hc-Myc mice, and of wildtype B6 mice (B6 wt, n = 5). Depicted are total cell numbers of the lymphoid organs as well as B cell numbers (B220⁺) and T cell numbers (CD3⁺). Total cell numbers were counted using a Neubauer chamber. The percentage of B and T cells was determined by flow cytometric analysis. Mean values are indicated in the graph with black lines and shown at the top of the symbols. Where ever small differences were observed, they were not significant.</p

Targeting strategy for homologous recombination in ES cells.

<p>The endogenous murine <i>c-Myc</i> gene was replaced with a humanized <i>c-Myc</i> gene (<i>hc-Myc</i>). <i>hc-Myc</i> has human DNA sequences from the first translation start side (CUG) up to 69 bp downstream of the stop codon (TAA). Non-coding sequences in exon 1 and exon 3 are of murine origin so that human c-MYC is expressed under the control of murine regulatory elements. (A) Endogenous murine <i>c-Myc</i> with its three exons (E1, E2, E3) and the three promoters (P1, P2, P3). Indicated are the two start codons (CUG, AUG) and the stop codon (TAA), as well as the flanking <i>EcoR</i>I restriction sites. (B) Murine <i>c-Myc</i> locus after homologous recombination with the targeting vector. Indicated in black are human sequences, in grey murine sequences and with triangles the two loxP-sites that flank the Neomycin-Geneticin resistance gene. (C) Recombined murine <i>c-Myc</i> locus after Cre-mediated deletion of the Neomycin-Geneticin resistance gene. A piece of mouse chromosome 15 (position 61.985.920 to 61.989.995) is replaced by a piece of human chromosome 8 (position 128.748.840 to 128.753.273). Dotted lines indicate recombination events. This schematic view displays <i>EcoR</i>I restriction sites, length of fragments after <i>Eco</i>RI-digestion, the sequence in the junction region between human and murine elements and the regions where the Southern blot probes (5′ probe and 3′ probe) bind. Cre  =  Cre recombinase.</p