Efficient male and female germline transmission of a human chromosomal vector in mice

Abstract

A small accessory chromosome that was mitotically stable in human fibroblasts was transferred into the hprt(-) hamster cell line CH and developed as a human chromosomal vector (HCV) by the introduction of a selectable marker and the 3' end of an HPRT minigene preceded by a loxP sequence. This HCV is stably maintained in the hamster cell line. It consists mainly of alphoid sequences of human chromosome 20 and a fragment of human chromosome region 1p22, containing the tissue factor gene F3. The vector has an active centromere, and telomere sequences are lacking. By transfecting a plasmid containing the 5' end of HPRT and a Cre-encoding plasmid into the HCV(+) hamster cell line, the HPRT minigene was reconstituted by Cre-mediated recombination and expressed by the cells. The HCV was then transferred to male mouse R1-ES cells and it did segregate properly. Chimeras were generated containing the HCV as an independent chromosome in a proportion of the cells. Part of the male and female offspring of the chimeras did contain the HCV. The HCV(+) F1 animals harbored the extra chromosome in >80% of the cells. The HCV was present as an independent chromosome with an active centromere and the human F3 gene was expressed from the HCV in a human-tissue-specific manner. Both male and female F1 mice did transmit the HCV to F2 offspring as an independent chromosome with properties similar to the original vector. This modified small accessory chromosome, thus, shows the properties of a useful chromosomal vector: It segregates stably as an independent chromosome, sequences can be inserted in a controlled way and are expressed from the vector, and the HCV is transmitted through the male and female germline in mice.

Figure 1

Modification and characterization of the small accessory chromosome (SAC). (A) Structure of the different vectors and strategy for introduction of new sequences into the SAC by Cre-mediated recombination. SAC sequences are indicated with a thick line, vector sequences with a thin line, and loxP sequences with an arrowhead. Neo, neomycine resistance gene driven by a thymidine kinase promoter; hyg , hygromycin resistance cassette driven by the PGK promoter; 5′- and 3′HPRT, human HPRT minigene driven by the SV40 early promoter; P, PstI cleavage site; B, BamHI cleavage site. Fragments used as a probe for Southern blot hybridizations are indicated with a shaded bar (not drawn to scale). (B) FISH with lissamine-labeled human CotI DNA (red signal) and biotine-labeled pBS-Neo/loxP/3′HPRT plasmid (green signal) on a methaphase of hybrid E10B1. The metaphase was counterstained with DAPI. (BI) Pseudocolored image, the circle shows the SAC; (BII) G-like banding derived from the DAPI channel. (C) In the last lane, inter-alu PCR products using E10B1 genomic DNA as a template are shown. Hamster and human genomic DNA were used as a negative and positive control, respectively. (D) FISH with biotin-labeled inter-alu PCR products on a metaphase of the human HT1080 cell line in which the SAC was introduced by MMCT. The circle indicates the SAC; arrowheads show the signals present on chromosome region 1p. (E) FISH with a FITC-(C₃TA₂)₃ peptide nucleic acid probe (green signals) and a lissamine-labeled α-satellite 2 probe (red signals) on metaphase spreads of the SAC⁺ HT 1080 cell line did not show a green signal on the SAC (circle). (F) Magnification of the SAC shown in E without the red signal of the α-satellite 2 probe.

Figure 1

Modification and characterization of the small accessory chromosome (SAC). (A) Structure of the different vectors and strategy for introduction of new sequences into the SAC by Cre-mediated recombination. SAC sequences are indicated with a thick line, vector sequences with a thin line, and loxP sequences with an arrowhead. Neo, neomycine resistance gene driven by a thymidine kinase promoter; hyg , hygromycin resistance cassette driven by the PGK promoter; 5′- and 3′HPRT, human HPRT minigene driven by the SV40 early promoter; P, PstI cleavage site; B, BamHI cleavage site. Fragments used as a probe for Southern blot hybridizations are indicated with a shaded bar (not drawn to scale). (B) FISH with lissamine-labeled human CotI DNA (red signal) and biotine-labeled pBS-Neo/loxP/3′HPRT plasmid (green signal) on a methaphase of hybrid E10B1. The metaphase was counterstained with DAPI. (BI) Pseudocolored image, the circle shows the SAC; (BII) G-like banding derived from the DAPI channel. (C) In the last lane, inter-alu PCR products using E10B1 genomic DNA as a template are shown. Hamster and human genomic DNA were used as a negative and positive control, respectively. (D) FISH with biotin-labeled inter-alu PCR products on a metaphase of the human HT1080 cell line in which the SAC was introduced by MMCT. The circle indicates the SAC; arrowheads show the signals present on chromosome region 1p. (E) FISH with a FITC-(C₃TA₂)₃ peptide nucleic acid probe (green signals) and a lissamine-labeled α-satellite 2 probe (red signals) on metaphase spreads of the SAC⁺ HT 1080 cell line did not show a green signal on the SAC (circle). (F) Magnification of the SAC shown in E without the red signal of the α-satellite 2 probe.

Figure 2

Southern analysis of the Cre-mediated introduction of a plasmid into the HCV. The pBS-Hyg/SV40 5′HPRT/loxP plasmid was integrated into the HCV by cotransfection with the Cre-encoding plasmid pOG231. Correct integration reconstitutes an HPRT minigene. Southern blots with DNA isolated from 10 independent HAT-resistant clones digested with either PstI or BamHI (lane 1–10) or untransfected E10B1 cells (lane C) were hybridized with probes for the hygromycin gene (upper panel), the 5′ end of the HPRT minigene (middle panel) and the 3′ end of the HPRT minigene (lower panel), respectively. A scheme of the probes and of the expected signals is shown in Fig. 1A.

Figure 3

Characterization of the HCV in F1 transchromosomal mice. Metaphase spreads of tail fibroblasts of HCV⁺ F1 mice were used for FISH. A cohybridization was performed with a biotinylated mouse Cot1 DNA probe (detected in green) and a lissamin-labeled human CotI DNA probe (red). (A) The green channel detecting the mouse sequences; (B) The red (human Cot1) and blue (DAPI counterstain) channels. (C,D) The detection of major and minor mouse satellite sequences (biotinylated, detected in green) and of the HCV (lissamin-labeled human Cot1 DNA, red), respectively. The left panels show the green channel; on the right the composite images are shown. Next, a codetection was performed of the HCV using a biotine-labeled alphoid 2 probe (green) and of CENP-C proteins by immunostaining (red signal). (E) The composite image (DAPI counterstain). The red CENP-C signal on the HCV is hidden by the strong green alphoid 2 signal. (F) The red channel only, of the image shown in E. Finally, a slide was sequentially hybridized with a FITC-labeled peptide nucleic acid telomere probe (G, green signals) and a lissamin-labeled human alphoid 2 probe (H, red signal). The poor quality of the metaphase in H results from the combination of the different protocols used for FISH with a peptide nucleic acid probe and a DNA probe. A circle shows the position of the HCV.

Figure 4

Tissue distribution of the HCV. Southern blot analysis of the HCV. DNA prepared from different tissues of an HCV⁺ F1 mouse was digested with XbaI, size-separated, and blotted. The left panel shows hybridization with a human alphoid-2 probe. The signal obtained for the different tissues is identical to the signal obtained for the E10B1 clone. The right panel shows the ethidium bromide–stained agarose gels.

Figure 5

Expression of human F3 in HCV⁺F1 and HCV⁺F2 mouse tissues. RNA was isolated from different tissues of a male (I) and a female (II) HCV⁺ F1 or HCV⁺ F2 mouse and brain of a normal control mouse. RT-PCR assays were developed detecting specifically human or mouse F3 mRNA. Equal amounts of cDNA were used for 30 cycles of PCR with the human F3 primers (hTF panels) or with the mouse F3 primers (mTF panels). A human fetal brain control is shown in lane C₁, lane C₂ shows a normal mouse brain control. (A) RT-PCR experiments with cDNA derived from tissues of transchromosomal F1 and F2 mice. B, brain; k, kidney; l, liver; I, intestine; m, muscle; n, negative control. (B) Western blot with 25 μg of total kidney proteins extracted from human kidney (hu), kidneys of four transchromosomal mice (lanes F1 I, F1 II, F2 I, and F2 II, respectively) and a normal mouse (m), stained with rabbit antihuman F3. (C) Immunostaining with rabbit anti human F3 of kidney from an HCV⁺ F1 mouse and an HCV⁻ littermate as a control shows positivity of the epithelia of the glomerulus, a typical human expression pattern only in the HCV⁺ kidney. The lower panel shows a human control kidney.