Gene Therapy (2002) 9, 708–712 2002 Nature Publishing Group All rights reserved 0969-7128/02 $25.00 www.nature.com/gt SESSION IV – CHROMOSOME ENGINEERING The use of chromosome-based vectors for animal transgenesis Y Kuroiwa1, H Yoshida1, T Ohshima1, T Shinohara2, A Ohguma1, Y Kazuki2, M Oshimura2, I Ishida1 and K Tomizuka1 1Pharmaceutical Research Laboratory, Kirin Brewery Co Ltd, Gunma, Japan; and 2Department of Molecular and Cell Genetics, School of Life Sciences, Faculty of Medicine, Tottori University, Tottori, Japan This article summarizes our efforts to use chromosome- human artificial chromosomes (HACs) containing a defined based vectors for animal transgenesis, which may have a chromosomal region should prevent the introduction of benefit for overcoming the size constraints of cloned trans- additional genes other than genes of interest and thus genes in conventional techniques. Since the initial trial for enhance the utility of chromosome vector system. Using this introducing naturally occurring human chromosome frag- technique a panel of HACs harboring inserts ranging in size ments (hCFs) with large and complex immunogulobulin (Ig) from 1.5 to 10 Mb from three human chromosomes (hChr2, loci into mice we have obtained several lines of trans-chro- 7, 22) has been constructed. Tc animals containing the mosomic (Tc) mice with transmittable hCFs. As expected the HACs may be valuable not only as a powerful tool for normal tissue-specific expression of introduced human functional genomics but also as an in vivo model to study genes was reproduced in them by inclusion of essential therapeutic gene delivery by HACs. remote regulatory elements. Recent development of ‘chro- Gene Therapy (2002) 9, 708–712. DOI: 10.1038/sj/gt/3301754 mosome cloning’ technique that enable construction of Keywords: chromosome; vector; transgenic animal; ES cells; Cre-loxP; telomere Introduction host cells, which may prevent artifacts and gene silenc- ing3 caused by multiple copy insertion of transgenes; (4) The ability of human chromosome-derived natural frag- there is no risk for insertional mutagenesis because they ments (hCFs) as vectors for introducing large stretches of are freely segregating from host chromosomes. 1 human DNA into mice was first demonstrated in 1997. On the other hand, one major problem of hCF vectors Transferred hCFs were stably maintained as an extra has been that they are structurally undefined.4,5 The hCFs chromosome in the somatic cells of mice and the human are usually generated as a consequence of accidental frag- genes included in them were expressed under proper mentation of intact chromosome during the chromosome tissue-specific regulation. In some cases they could be transfer process and run the risk of containing unrelated, transmitted through the mouse germline, resulting in the deleterious genes in addition to the genes of interest. In establishment of novel mouse strains (trans-chromosomic this context, we recently developed a ‘chromosome clon- 1,2 (Tc) mice) containing a heritable hCF. Thus, such an ing’ technique that enabled construction of human arti- approach employing chromosome vectors for animal ficial chromosomes (HACs) including only defined transgenesis has been thought to be useful for overcom- chromosomal regions in the homologous recombination ing the size constraints of cloned transgenes in conven- proficient chicken DT40 cell line.6 The complete human tional techniques and facilitate functional studies of genome sequence which is now available,7 should facili- human genome in the ‘post sequencing’ era. tate the minimization of HACs containing only desired Compared with conventional techniques using cloned chromosomal regions. transgenes, the chromosome vector system have several Here, we outline the current status of our ‘trans- advantages: (1) whole genomic sequences including all chromosomic’ technology and the recent progress on the the introns and essential regulatory elements can be used development of HACs. as transgenes, which results in the correct and controlled expression of transgenes in vivo; (2) very large genes, gene clusters and specific chromosomal regions, which Results and discussion cannot be cloned as contiguous DNA fragments by clon- ing techniques, can be introduced; (3) chromosome vec- Trans-chromosomic mice tors are maintained as a single copy chromosome in the Our procedure to introduce chromosome vectors into mice is illustrated in Figure 1. Microcell-mediated chro- mosome transfer (MMCT) was employed to introduce Correspondence: K Tomizuka, Pharmaceutical Research Laboratory, Kirin chromosome vectors into mouse embryonic stem (ES) Brewery Co Ltd, 3 Miyahara-cho, Takasaki-shi, Gunma, 370-1295, Japan cells.1 Our previous studies indicated that the use of Chromosome-based vectors for animal transgenesis Y Kuroiwa et al 709 Figure 1 A schematic diagram showing the construction of MH(ES) cells to produce chimeric mice and Tc mice expressing human genes on the transferred hCF. G418r, G418-resistant. small human chromosome fragments (hCFs) may addition to the danger of including unrelated, deleterious increase the probability of successful chimera production genes as mentioned above. We therefore developed a from microcell hybrid ES (MH(ES)) cells and germline ‘chromosome-cloning’ technique where a defined transmission of the hCFs.2 Therefore, the library compris- chromosomal region could be cloned into a stable human ing approximately 700 independent clones of human– minichromosome vector by combination of Cre/loxP- mouse monochromosomal hybrids was screened to mediated chromosome translocation13 and telomere- obtain small hCFs containing the gene of interest.1,2 To directed chromosome truncation8 in homologous recom- date we successfully produced chimeric mice from bination-proficient chicken DT40 cells.14 MH(ES) cells containing the hCF derived from hChr2, 4, In our system (Figure 2), the hCF(SC20) was chosen as 6, 7, 11, 14, 21 or 22.1,2,8,9 In addition, germline trans- a basal minichromorome vector (SC20 vector) because of mission of transferred hCF was observed in chimeras its relatively high mitotic and meiotic stability in mice.2,12 produced from MH(ES) cells containing the hChr2, 7, 14 At first, a loxP sequence was integrated to RNR2 locus or 21-derived hCF.1,2,10 on the SC20 vector in DT40 cells, to which various Functional expression of introduced genes was exten- chromosomal regions could be cloned. As the first model sively investigated in the Tc mice containing the transmit- of HAC construction carrying a defined chromosomal table hChr2-derived hCF (hCF(2-W23)) and hChr14- insert, we attempted to clone the region between HCF2 derived hCF (hCF(SC20)), each of which include and LIF loci containing Ig␭ locus on the hChr22, the 10 immunoglobulin (Ig) kappa light chain (2 Mb) and heavy Mb-sized hChr22-derived insert. In DT40 cells, the chain (1.5 Mb) locus, respectively.1,2 The results of struc- hChr22 was truncated at the LIF locus by integrating tural analysis of human Ig mRNA and Ig protein human telomeric repeats (TTAGGG)n, followed by inte- expression in the sera of Tc mice suggested that these gration of a loxP sequence to the HCF2 locus.6 Next, the large and complex loci were properly expressed to recon- DT40 clone containing the SC20 vector and the DT40 stitute the diverse and functional repertoire of human Igs clone containing the hChr22 fragment (hCF(22)) were in the Tc mice.1 Furthermore, the introduction of fused, resulting in DT40 cell hybrids with both SC20 vec- hCF(SC20) into endogenous IgH-knockout strain, in tor and hCF(22). The hybrids were transfected with a Cre which functional B-lymphocytes and Ig production are recombinase-expression vector to induce Cre/loxP- absent, by mating resulted in the rescue of its defects, mediated chromosomal translocation between the SC20 indicating that the stability and functionality of the vector and hCF(22). Occurrence of the translocation was hCF(SC20) is likely to be sufficient for restoration of B confirmed by nested PCR analysis and then the hybrid cells in adult mice.2 cells containing the possible translocated chromosomal Stability tests using MH(ES) cell lines2,6revealed the fragment were isolated by fluorescence activated cell hCF(SC20) was highly stable (Ͻ0.1% loss/doubling) in sorter (FACS).6 Fluorescence in situ hybridization (FISH) contrast to the hCF(2-W23) (3.2% loss/doubling), hCF(22) analysis in the sorted cells demonstrated successful gen- (>5% loss/doubling) and the hChrY-derived minichro- eration of the human artificial chromosome (HAC) carry- mosome reported by Shen et al.11 Similar results were ing the defined hChr22-derived insert, named ␭HAC obtained in various types of cell lines from different spec- (Figure 3). ies12 and in the somatic and germ cells of Tc mice.2 Thus, To see the functionality of the HAC, we transferred the it was suggested that each hCF may have an intrinsic ␭HAC to mouse embryonic stem (ES) cells where level of mitotic and meiotic stability, which is less affec- endogenous IgH and Ig␬ genes were inactivated. In the ted by the type of host cells. ES cells, whereas hCF(22) was drastically lost after 15- day culture (about 20 doublings) under nonselective con- Construction of HACs dition, ␭HAC carrying this unstable hCF(22)-derived This differential stability of hCFs could be another prob- insert, as well as the SC20 vector itself, was stably main- lem in using the hCFs as gene delivery vectors, in tained at 99.8% retention per cell division throughout the Gene Therapy Chromosome-based vectors for animal transgenesis Y Kuroiwa et al 710 Figure 2 Schematic diagram of the chromosome-cloning system. DT40/SC20 vector, the DT40 clone with the SC20 vector where a loxP sequence is integrated at the RNR2 locus. DT40/hCF22, the DT40 clone with the hChr22 fragment truncated at the LIF locus where a loxP sequence is integrated at the HCF2 locus. The two DT40 clones were fused, resulting in the hybrid clone containing both SC20 vector and hCF22.