P1 Artificial Chromosome Research Articles

T(14;22)(q32;q11) in non‐Hodgkin lymphoma and myeloid leukaemia: molecular cytogenetic investigations

Two non-Hodgkin lymphomas (NHL), one chronic lymphocytic leukaemia/small lymphocytic lymphoma and one diffuse large B-cell lymphoma and three cases of myeloid leukaemia, two chronic (CML) and one acute (AML), showed, by G-banding analysis, apparently identical chromosomal translocations t(14;22)(q32;q11), in three of the cases as the sole abnormality. Fluorescence in situ hybridisation (FISH) analysis with locus-specific probes for ABL at 9q34 [bacterial artificial chromosomes (BACs) 835J22 and 1132H12], IGH at 14q32 [P1 artificial chromosome (PAC) 998D24] and IGL (PAC 1019H10) and BCR (BAC 74M14) at 22q11, as well as multicolour in situ hybridisation (M-FISH) analyses were performed. A three-way variant translocation of the classical t(9;22)(q34;q11), t(9;22;14)(q34;q11;q32), involving both BCR and ABL, was unravelled by the molecular cytogenetic investigations in the three myeloid leukaemia cases; a similar variant translocation has previously been reported in seven CML. The two cases of NHL (one NHL with a similar 14;22-translocation has been reported previously) had no involvement of BCR or ABL, but instead the IGH and IGL genes were shown to be juxtaposed by the t(14;22)(q32;q11). How such a rearrangement with recombination of IGH and IGL might elicit a pathogenetic effect is completely unknown.

Delineation of a supernumerary marker chromosome utilizing a multimodal approach of G‐banding, fluorescent in situ hybridization, confirmatory P1 artificial chromosome fluorescent in situ hybridization, and high‐resolution comparative genomic hybridization

We describe the structure of a supernumerary marker in a child who presented with a right atretic ear and multiple congenital anomalies. Using G-banding, fluorescent in situ hybridization (FISH), P1 artificial chromosome FISH and high-resolution comparative genomic hybridization (CGH), the marker was demonstrated to be a derivative chromosome resulting from malsegregation of a paternal 8;22 translocation: 47,XY, +der(22)t(8;22)(q24.1; q11.2). This case is noteworthy because the marker, while sharing similarities to der(22) in the Cat Eye syndrome (CES), also contains chromosome 8q material. This partial 8q trisomy confounds the diagnosis of CES associated with pure trisomy or pure tetrasomy 22q. The paternal translocation is noted with prolonged infertility and oligospermia, which again highlights the utility and necessity of chromosome analysis in this setting.

From Mapping to Sequencing, Post-sequencing and Beyond

The Rice Genome Research Program (RGP) in Japan has been collaborating with the international community in elucidating a complete high-quality sequence of the rice genome. As the pioneer in large-scale analysis of the rice genome, the RGP has successfully established the fundamental tools for genome research such as a genetic map, a yeast artificial chromosome (YAC)-based physical map, a transcript map and a phage P1 artificial chromosome (PAC)/bacterial artificial chromosome (BAC) sequence-ready physical map, which serve as common resources for genome sequencing. Among the 12 rice chromosomes, the RGP is in charge of sequencing six chromosomes covering 52% of the 390 Mb total length of the genome. The contribution of the RGP to the realization of decoding the rice genome sequence with high accuracy and deciphering the genetic information in the genome will have a great impact in understanding the biology of the rice plant that provides a major food source for almost half of the world's population. A high-quality draft sequence (phase 2) was completed in December 2002. Since then, much of the finished quality sequence (phase 3) has become available in public databases. With the completion of sequencing in December 2004, it is expected that the genome sequence would facilitate innovative research in functional and applied genomics. A map-based genome sequence is indispensable for further improvement of current rice varieties and for development of novel varieties carrying agronomically important traits such as high yield potential and tolerance to both biotic and abiotic stresses. In addition to genome sequencing, various related projects have been initiated to generate valuable resources, which could serve as indispensable tools in clarifying the structure and function of the rice genome. These resources have been made available to the scientific community through the Rice Genome Resource Center (RGRC) of the National Institute of Agrobiological Sciences (NIAS) to enable rapid progress in research that will lead to thorough understanding of the rice plant. As the next trend in rice genome research will focus on determining the function of about 40,000-50,000 genes predicted in the genome as well as applying various genomics tools in rice breeding, an unlimited access to rice DNA and seed stocks will provide a broad community of scientists with the necessary materials for formulating new concepts, developing innovative research and making new scientific discoveries in rice genomics.

Multiple Promoters Direct Expression of Three AKAP12 Isoforms with Distinct Subcellular and Tissue Distribution Profiles

A Kinase Anchoring Protein 12 (AKAP12; also known as src-suppressed C kinase substrate (SSeCKS) and Gravin) is a multivalent anchoring protein with tumor suppressor activity. Although expression of AKAP12 has been examined in a number of contexts, its expression control remains to be elucidated. Herein, we characterize the genomic organization of the AKAP12 locus, its regulatory regions, and the spatial distribution of the proteins encoded by the AKAP12 gene. Using comparative genomics and various wet-lab assays, we show that the AKAP12 locus is organized as three separate transcription units that are governed by non-redundant promoters coordinating distinct tissue expression profiles. The proteins encoded by the three AKAP12 isoforms (designated alpha, beta, and gamma) share >95% amino acid sequence identity but differ at their N termini. Analysis of the targeting of each isoform reveals distinct spatial distribution profiles. An N-terminal myristoylation motif present in AKAP12alpha is shown to be necessary and sufficient for targeted expression of this AKAP12 isoform to the endoplasmic reticulum, a novel subcellular compartment for AKAP12. Our results demonstrate heretofore unrecognized complexity within the AKAP12 locus and suggest a mechanism for genetic control of signaling specificity through distinct regulation of alternately targeted anchoring protein isoforms.

Pericentric chromosome 8 inversion associated with the 5?RUNX1/3?CBFA2T1 gene in acute myeloid leukemia cases

In the present paper we report pericentric chromosome 8 inversions in two (2.4%) of 82 acute myeloid leukemia (AML) cases characterized by the 5'RUNX1/3'CBFA2T1 fusion gene. Molecular cytogenetic characterization was achieved using appropriate bacterial artificial chromosome (BAC) and P1 artificial chromosome (PAC) probes in fluorescence in situ hybridization (FISH) experiments. In these two cases the fusion gene was detected on the der(8) short arm, resulting from a pericentric chromosome 8 inversion followed by a t(8;21) rearrangement. These results suggest that heterogeneous mechanisms can lead to the generation of the 5'RUNX1/3'CBFA2T1chimeric gene.

Familial 22q11.2 deletions in DiGeorge/velocardiofacial syndrome are predominantly smaller than the commonly observed 3Mb

Purpose: DiGeorge/velocardiofacial syndrome (DG/VCFS) is the most common cytogenetically characterized microdeletion of 22q11.2 region. In ≈90% of patients, the deletion size is 3 Mb, whereas the remaining range from 1.5 to 2.5 Mb. The purpose of this study was to test the hypothesis that small deletions may be more easily tolerated in a familial fashion than larger deletions, especially for this syndrome.Method: Sixteen FISH probes designed from bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) mapped to 22q11.2 were used to determine the deletion sizes in 22 individuals from ten families with familial 22q11.2 deletion detected by standard FISH tests.Result: Seven families had deletions of < 3 Mb (≈1.5 Mb) in size and 3 families had the common 3-Mb deletion. The 70% frequency of smaller sized deletions among this group of patients with familial del(22)(q11.2) is significantly higher than that reported among unselected group of patients with del(22)(q11.2) (P < 0.0001, Fisher exact test).Conclusion: Familial del(22)(q11.2) are predominantly smaller than the common deletion size of 3 Mb, indicating that there may be some underlying mechanisms that favor parent-to-child transmission of smaller deletions in individuals with del(22)(q11.2), therefore, underscoring the need to exclude a familial basis in cases of del(22)(q11.2) smaller than 3 Mb.

Cloning and reporter analysis of human mitochondrial phospho enolpyruvate carboxykinase gene promoter

Phosphoenolpyruvate carboxykinase (PEPCK) is one of the key regulatory enzymes in gluconeogenesis. In human liver, PEPCK is about equally distributed in both cytosol (PEPCK-1) and mitochondria (PEPCK-2). The human pepck2 gene and cDNA have been reported, but the cloning of the promoter region of the pepck2 gene has not been elucidated yet. We isolated and characterized human genomic P1-artificial chromosome (PAC) clones carrying the human pepck2 gene promoter. The oligocapping method revealed that the transcriptional start point (tsp) of the human pepck2 gene is located at 97 bp upstream of the first adenine residue of the translation start site. We also determined the nucleotide sequence to 1819 bp upstream of tsp. Sequence analysis of this region revealed that it contained several potential regulatory elements, including five GC boxes and three CCAAT boxes. Reporter analysis using transient transfection with firefly luciferase synthetic gene indicated 5′ flanking region up to 822 bp, and 317 bp upstream of tsp had transcriptional activity. These results suggest that these regions of the human pepck2 gene play an important role for its expression.

Insertions generating the 5'RUNX1/3'CBFA2T1 gene in acute myeloid leukemia cases show variable breakpoints.

Translocation t(8;21)(q22;q22) is a common karyotypic abnormality detected in about 15% of acute myeloid leukemia (AML) cases. The rearrangement results in fusion of the RUNX1 (also known as AML1) and CBFA2T1 (also known as ETO) genes, generating a 5'RUNX1/3'CBFA2T1 transcriptionally active fusion gene on derivative chromosome 8, but some cases with ins(21;8) and ins(8;21) have been observed. However, a detailed breakpoint characterization of the insertion events has never been reported. In the present article, we describe six insertion events among 82 (7.3%) AML cases characterized by the RUNX1/CBFA2T1 fusion. Using FISH experiments with appropriate bacterial artificial chromosome (BAC) and P1 artificial chromosome (PAC) probes, we were able to perform a detailed molecular cytogenetic characterization of one case with ins(8;21) and five with ins(21;8). Our analysis revealed that insertions generating the 5'RUNX1/3'CBFA2T1 gene showed variable breakpoints; the size of the inserted elements ranged from 2.4 to 44 Mb.

Cytogenetic and molecular cytogenetic findings in 43 aneurysmal bone cysts: aberrations of 17p mapped to 17p13.2 by fluorescence in situ hybridization

Aneurysmal bone cyst is a benign, cystic lesion of bone composed of blood-filled spaces separated by fibrous septa. Relatively few cases of aneurysmal bone cyst have been cytogenetically characterized, yet abnormalities of the short arm of chromosome 17 appear to be recurrent. In this study, conventional cytogenetic analysis of 43 aneurysmal bone cyst specimens from 38 patients over a 12-year period revealed clonal chromosomal abnormalities in 12 specimens. Karyotypic anomalies of 17p, including a complex translocation and inversion, were identified in eight of these 12 specimens. In an effort to further define the aberrant 17p breakpoint, fluorescence in situ hybridization (FISH) analyses were performed using a series of probe combinations spanning a 5.1 Mb region between the TP53 (17p13.1) and Miller–Dieker lissencephaly syndrome (17p13.3) gene loci. These studies revealed the critical breakpoint locus at 17p13.2, flanked proximally by an RP11-46I8, RP11-333E1, and RP11-457I18 bacterial artificial chromosome (BAC) probe cocktail and distally by an RP11-198F11 and RP11-115H24 BAC and RP5-1050D4 P1 artificial chromosome (PAC) probe cocktail. Overall, abnormalities of the 17p13.2 locus were identified by metaphase and/or interphase cell FISH analysis in 22 of 35 (63%) aneurysmal bone cyst specimens examined including 26 karyotypically normal specimens. These cytogenetic and molecular cytogenetic findings expand our knowledge of chromosomal alterations in aneurysmal bone cyst, further localize the critically involved 17p breakpoint, and provide an alternative approach (ie FISH) for detecting 17p abnormalities in nondividing cells of aneurysmal bone cysts. The latter could potentially be utilized as an adjunct in diagnostically challenging cases.

Integrated and sequence-ordered BAC- and YAC-based physical maps for the rat genome.

As part of the effort to sequence the genome of Rattus norvegicus, we constructed a physical map comprised of fingerprinted bacterial artificial chromosome (BAC) clones from the CHORI-230 BAC library. These BAC clones provide approximately 13-fold redundant coverage of the genome and have been assembled into 376 fingerprint contigs. A yeast artificial chromosome (YAC) map was also constructed and aligned with the BAC map via fingerprinted BAC and P1 artificial chromosome clones (PACs) sharing interspersed repetitive sequence markers with the YAC-based physical map. We have annotated 95% of the fingerprint map clones in contigs with coordinates on the version 3.1 rat genome sequence assembly, using BAC-end sequences and in silico mapping methods. These coordinates have allowed anchoring 358 of the 376 fingerprint map contigs onto the sequence assembly. Of these, 324 contigs are anchored to rat genome sequences localized to chromosomes, and 34 contigs are anchored to unlocalized portions of the rat sequence assembly. The remaining 18 contigs, containing 54 clones, still require placement. The fingerprint map is a high-resolution integrative data resource that provides genome-ordered associations among BAC, YAC, and PAC clones and the assembled sequence of the rat genome.

Differential expression of the closely linked KISS1, REN, and FLJ10761 genes in transgenic mice.

We previously reported the development and characterization of transgenic mice containing a large 160-kb P1 artificial chromosome (PAC) encompassing the renin (REN) locus from human chromosome 1. Here we demonstrate that PAC160 not only encodes REN, but also complete copies of the next upstream (KISS1) and downstream (FLJ10761) gene along human chromosome 1. Incomplete copies of the second upstream (PEPP3) and downstream (SOX13) genes are also present. The gene order PEPP3-KISS1-REN-FLJ10761-SOX13 is conserved in mice containing either one or two copies of the REN locus. Despite the close localization of KISS1, REN, and FLJ10761, they each exhibit distinct, yet overlapping tissue-specific expression profiles in humans. The tissue-specific expression patterns of REN and FLJ10761 were retained in transgenic mice containing PAC160. Expression of REN and FLJ10761 were also proportional to copy number. Expression of KISS1 in PAC160 mice showed both similarities and differences to humans. These data suggest that expression of gene blocks encoded on large genomic clones are retained when the clones are used to generate transgenic mice. Genomic elements which act to insulate genes from their neighbors are also apparently retained.

Mitochondrial transcription factor A regulates mtDNA copy number in mammals.

Mitochondrial DNA (mtDNA) copy number regulation is altered in several human mtDNA-mutation diseases and it is also important in a variety of normal physiological processes. Mitochondrial transcription factor A (TFAM) is essential for human mtDNA transcription and we demonstrate here that it is also a key regulator of mtDNA copy number. We initially performed in vitro transcription studies and determined that the human TFAM protein is a poor activator of mouse mtDNA transcription, despite its high capacity for unspecific DNA binding. Next, we generated P1 artificial chromosome (PAC) transgenic mice ubiquitously expressing human TFAM. The introduced human TFAM gene was regulated in a similar fashion as the endogenous mouse Tfam gene and expression of the human TFAM protein in the mouse did not result in down-regulation of the endogenous expression. The PAC-TFAM mice thus had a net overexpression of TFAM protein and this resulted in a general increase of mtDNA copy number. We used a combination of mice with TFAM overexpression and TFAM knockout and demonstrated that mtDNA copy number is directly proportional to the total TFAM protein levels also in mouse embryos. Interestingly, the expression of human TFAM in the mouse results in up-regulation of mtDNA copy number without increasing respiratory chain capacity or mitochondrial mass. It is thus possible to experimentally dissociate mtDNA copy number regulation from mtDNA expression and mitochondrial biogenesis in mammals in vivo. In conclusion, our results provide genetic evidence for a novel role for TFAM in direct regulation of mtDNA copy number in mammals.

Microinjection of BAC DNA into the pronuclei of fertilized mouse oocytes.

AbstractMicroinjection of DNA into the pronuclei of fertilized oocytes is one of the two most commonly used methods for gene transfer into the mouse genome (1,2). The first successful attempt to perform this technique was carried out by Lin in 1966 (3), who could show that the early fertilized embryo could survive the mechanical damage of inserting a glass needle into the pronucleus. However, it was not until 1981 that small DNA fragments were integrated into the genome (4). This technique is well described, and has now become a standard procedure (5,6). More recently, the use of larger DNA fragments has been established. Yeast artificial chromosome (YAC) (7,8), P1 artificial chromosome (PAC) (9,10), and bacterial artificial chromosome (BAC) DNA (11,12) can all be used for the generation of transgenic mice. A comprehensive review of BAC and YAC transgenesis is given by Giraldo and Montoliu (13) extensively comparing these applications. Although the basic technique for microinjection of large constructs are similar to those used for shorter DNA segments, there are some special requirements (14). In this chapter, we describe relevant steps for microinjection using large DNA constructs.KeywordsBacterial Artificial ChromosomeEmbryo TransferYeast Artificial ChromosomeInjection NeedleBunsen BurnerThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The effects of plasmid copy number and sequence context upon transfection efficiency

It is known that large P1 artificial chromosome (PAC) vectors exhibit reduced transfection efficiency in comparison to small plasmid vectors. We investigated the dynamics of this effect, by comparing expression from a small plasmid (4.7 kb) and a PAC vector (111 kb) containing the Enhanced Green Fluorescent Protein (EGFP) reporter gene under the control of a P CMV promoter. EGFP expression was detected by fluorescence activated cell sorting (FACS). We found that the lower transfection efficiency of PAC vectors represents both a smaller percentage of cells expressing the transgene, and a lower level of expression per cell. We have shown that the lower number of plasmid molecules administered per cell in a PAC transfection does not explain this effect, and that this effect does not act in trans. Surprisingly, dilution of a reporter construct with an irrelevant plasmid did not appear to compromise transfection efficiency; in fact, a dilution of 1/10 slightly enhanced transfection. Therefore, it seems that the plasmid content of a liposome–DNA complex need not be 100% reporter construct for optimum transfection efficiency. This discovery has potential practical utility in a number of applications.

The functional mapping of long-range transcription control elements of the HOX11 proto-oncogene

Mapping of transcriptional control elements normally depends on the generation of a series of deletion mutants. The consequences of particular deletions are then functionally assessed by their ability to alter gene expression. The information derived from such investigations provides a general regulatory profile of the gene of interest, as well as generating a focus for future experiments. Due to the limitations of conventional DNA cloning methods, it has previously not been possible to use such an approach to rapidly assess the role of long-range regulatory elements that frequently lie further than 20 kb away from the coding region. In order to identify regulatory elements of the proto-oncogene HOX11 that may be mutated in a subset of childhood T-cell acute lymphoblastic leukaemia specimens, we generated nested deletions from a P1 artificial chromosome (PAC). This clone contained 95 kilobases (kb) of the HOX11 locus at 10q24; including 63 kb of 5 ′ regulatory DNA. The deletion series was produced by the use of a recombination based cloning system and clones were subsequently transfected into mammalian cells. We have identified several long-range regulatory elements that mediate transcriptional control of HOX11. This approach is simple, rapid, and inexpensive. Furthermore, it generates multiple deletion clones in a single experiment. This novel approach opens up a new avenue for investigating long-range transcription control. Additionally, by allowing analysis of these elements in the natural context of large integrants the approach does not require the use of artificial extrachromosomal elements. This methodology can be applied to any gene cloned into a PAC or BAC vector and could also be useful in identifying appropriately sized deletion mutants for functional testing in transgenic models.

Mini-λ: a tractable system for chromosome and BAC engineering

The bacteriophage lambda (λ) recombination system Red has been used for engineering large DNA fragments cloned into P1 and bacterial artificial chromosomes (BAC or PAC) vectors. So far, this recombination system has been utilized by transferring the BAC or PAC clones into bacterial cells that harbor a defective λ prophage. Here we describe the generation of a mini-λ DNA that can provide the Red recombination functions and can be easily introduced by electroporation into any E. coli strain, including the DH10B-carrying BACs or PACs. The mini-λ DNA integrates into the bacterial chromosome as a defective prophage. In addition, since it retains attachment sites, it can be excised out to cure the cells of the phage DNA. We describe here the use of the mini-λ recombination system for BAC modification by introducing a selectable marker into the vector sequence of a BAC clone. In addition, using the mini-λ, we create a single missense mutation in the human BRCA2 gene cloned in a BAC without the use of any selectable marker. The ability to generate recombinants very efficiently demonstrates the usefulness of the mini-λ as a very simple mobile system for in vivo genome engineering by homologous recombination, a process named recombineering.

Transgenic targeting with regulatory elements of the humanCD34 gene

The human CD34 gene is expressed on early progenitor and stem cells in the bone marrow. Here we report the isolation of the human CD34 locus from a human P1 artificial chromosome (PAC) library and the characterization and evaluation of this genomic fragment for expression of reporter genes in stable cell lines and transgenic mice. We show that a 160-kb fragment spanning 110 kb of the 5' flanking region and 26 kb of the 3' flanking region of the CD34 gene directs expression of the human CD34 gene in the bone marrow of transgenic mice. The expression of human CD34 transgenic RNA in tissues was found to be similar to that of the endogenous murine CD34 gene. Colony-forming cell assays showed that bone marrow cells staining positive for human CD34 consist of early progenitor cells in which expression of CD34 decreased with cell maturation. In order to test the construct for its ability to express heterologous genes in vivo, we used homologous recombination in bacteria to insert the tetracycline-responsive transactivator protein tTA. Analysis of transgenic human CD34-tTA mice by cross breeding with a strain carrying Cre recombinase under control of a tetracycline-responsive element demonstrated induction of Cre expression in mice in a pattern consistent with the expression of the human CD34 transgene.

Evidence for a fast, intrachromosomal conversion mechanism from mapping of nucleotide variants within a homogeneous alpha-satellite DNA array.

Assuming that patterns of sequence variants within highly homogeneous centromeric tandem repeat arrays can tell us which molecular turnover mechanisms are presently at work, we analyzed the alpha-satellite tandem repeat array DXZ1 of one human X chromosome. Here we present accurate snapshots from this dark matter of the genome. We demonstrate stable and representative cloning of the array in a P1 artificial chromosome (PAC) library, use samples of higher-order repeats subcloned from five unmapped PACs (120-160 kb) to identify common variants, and show that such variants are presently in a fixed transition state. To characterize patterns of variant spread throughout homogeneous array segments, we use a novel partial restriction and pulsed-field gel electrophoresis mapping approach. We find an older large-scale (35-50 kb) duplication event supporting the evolutionarily important unequal crossing-over hypothesis, but generally find independent variant occurrence and a paucity of potential de novo mutations within segments of highest homogeneity (99.1%-99.3%). Within such segments, a highly nonrandom variant clustering within adjacent higher-order repeats was found in the absence of haplotypic repeats. Such variant clusters are hardly explained by interchromosomal, fixation-driving mechanisms and likely reflect a fast, localized, intrachromosomal sequence conversion mechanism.

Analysis of chromosome conservation in Lemur catta studied by chromosome paints and BAC/PAC probes.

A panel of human chromosome painting probes and bacterial and P1 artificial chromosome (BAC/PAC) clones were used in fluorescence in situ hybridization (FISH) experiments to investigate the chromosome conservation of the ring-tailed lemur (Lemur catta, LCA) with respect to human. Whole chromosome paints specific for human chromosomes 7, 9, 11, 13, 14, 17, 18, 20, 21, and X were found to identify a single chromosome or an uninterrupted chromosomal region in LCA. A large set of partial chromosome paints and BAC/PAC probes were then used to refine the characterization of the rearrangements differentiating the two karyotypes. The results were also used to reconstruct the ancestral Lemuridae karyotype. Lemur catta, indeed, can be used as an outgroup, allowing symplesiomorphic (ancestral) rearrangements to be distinguished from apomorphic (derived) rearrangements in lemurs. Some LCA chromosomes are difficult to distinguish morphologically. The 'anchorage' of most LCA chromosomes to specific probes will contribute to the standardization of the karyotype of this species.

Differential regulation of the human and murine CD34 genes in hematopoietic stem cells.

Human CD34 (hCD34)-positive cells are used currently as a source for hematopoietic transplantation in humans. However, in steady-state murine hematopoiesis, hematopoietic stem cells (HSCs) with long-term reconstitution activity are found almost exclusively in the murine CD34 (mCD34)-negative to low fraction. To evaluate the possible differences in hCD34 and mCD34 gene expression in hematopoiesis, we made transgenic mouse strains with human genomic P1 artificial chromosome clones spanning the entire hCD34 genomic locus. In all transgenic mouse strains, a vast majority of phenotypic and functional HSC populations including mCD34(-/lo) express the hCD34 transgene. These data strongly support the notion that hCD34(+) human bone marrow cells contain long-term HSCs that can maintain hematopoiesis throughout life.