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Genes to Cells (2004) 9, 1043-1053. doi:10.1111/j.1365-2443.2004.00788.x
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Locus control region elements HS2 and HS3 in combination with chromatin boundaries confer high-level expression of a human ß-globin transgene in a centromeric region

Sung-Hae Lee Kang1,{dagger}, Padraic P Levings1,{dagger}, Felicie Andersen1, Philip J Laipis1,4, Kenneth I Berns2,4,5, Robert T Zori3 and Jörg Bungert1,4,5,*

1 Department of Biochemistry and Molecular Biology
2 Department of Molecular Genetics and Microbiology
3 Department of Pediatrics
4 Powell Gene Therapy Center and Center for Mammalian Genetics
5 Genetics Institute, College of Medicine, University of Florida, Gainesville, Florida 32610, USA


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Expression constructs are subject to position-effects in transgenic assays unless they harbour elements that protect them from negative or positive influences exerted by chromatin at the site of integration. Locus control regions (LCRs) and boundary elements are able to protect from position effects by preventing heterochromatization of linked genes. The LCR in the human ß-globin gene locus is located far upstream of the genes and composed of several erythroid specific DNase I hypersensitive (HS) sites. Previous studies demonstrated that the LCR HS sites act synergistically to confer position-independent and high-level globin gene expression at different integration sites in transgenic mice. Here we show that LCR HS sites 2 and 3, in combination with boundary elements derived from the chicken ß-globin gene locus, confer high-level human ß-globin gene expression in different chromosomal integration sites in transgenic mice. Moreover, we found that the construct is accessible to nucleases and highly expressed when integrated in a centromeric region. These results demonstrate that the combination of enhancer, chromatin opening and boundary activities can establish independent expression units when integrated into chromatin.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The analysis of regulatory DNA elements in the context of transgenic mice is often complicated by the observation that expression constructs are subject to position of integration effects which causes variable expression levels in independent lines. Locus control regions (LCRs) are powerful genetic elements that are able to protect genes from position effects in transgenic assays (Grosveld et al. 1987; Li et al. 2002). LCRs are often composite elements containing multiple core regions that exhibit heightened sensitivity to nucleases in specific cell types (Tuan et al. 1985; Forrester et al. 1987; Bonifer 2000). These HS sites can be clustered or spread throughout a gene locus (Bonifer 2000; Li et al. 2002). Many studies have shown that the LCR HS sites of the human ß-globin gene locus synergize to confer high-level and position-independent expression (Bungert et al. 1995; Milot et al. 1996; Li et al. 1998; May et al. 2000; Molete et al. 2001). Results from genetic as well as conformational studies suggest that the HS sites of the ß-globin LCR interact with each other and with the genes they activate at a particular developmental stage (Bungert et al. 1995, 1999; Tolhuis et al. 2002; Carter et al. 2002). These interactions could establish a configuration that protects the genes from negative effects exerted by neighbouring chromatin (De Laat & Grosveld 2004). Another group of regulatory DNA elements that protect genes from position of integration effects are the so-called boundary elements. These elements were first discovered as sequences that protect transgenes from position effect variegation (PEV) in Drosophila (Kellum & Schedl 1991). Similar elements were discovered in higher eukaryotic cells. For example, matrix attachment regions (MARs) flanking the chicken lysozyme gene locus protect the gene from position effects in transgenic mice (Stief et al. 1989). Likewise, elements flanking the chicken ß-globin gene locus, cHS4, are able to protect reporter genes in transgenic assays (Chung et al. 1997; West et al. 2002). Chicken HS4 harbours two distinguishable activities; it blocks the function of enhancers on activating promoters, and it establishes a boundary between open and closed chromatin (West et al. 2002). While enhancer blocking activity of cHS4 is mediated by CTCF (Bell et al. 1999), proteins mediating the boundary function remain to be determined. Recent results suggest that some insulators may be tethered to nuclear compartments, e.g. nuclear pore complex or nucleolus, via interactions with proteins known to reside in these structures (Ishii et al. 2002; Yusufzai et al. 2004).

A promising strategy for avoiding position of integration effects is to direct the transgene into a specific site in the genome. Several strategies have been applied to directing transgenes into specific genomic sites (Bouhassira et al. 1997; Belteki et al. 2003; Porteus et al. 2003). In this study we utilized components of the adeno associated virus (AAV) integration machinery with the goal of directing a human ß-globin expression construct into a specific site in the mouse genome. AAV is a small human virus that is able to establish latent infection by integrating its DNA into a specific site on human chromosome 19, called the AAVS1 site (Kotin et al. 1992; Linden et al. 1996). Integration is mediated by DNA sequences present in the viral genome, as well as by the AAV encoded rep protein, which contains ATPase, helicase, and DNA-nicking activities (Weitzman et al. 1994; Ryan et al. 1996; Zhou et al. 1999). We generated transgenic mice containing the human AAVS1 integration site. The ß-globin expression construct contained sequences from AAV that have previously been shown to be critical or to enhance integration into the AAVS1 site (Philpott et al. 2002). This construct was incubated with recombinant AAV rep protein and the mixture injected into the pronuclei of fertilized murine oocytes transgenic for the AAVS1 site. Despite using different conditions and rep/DNA molar ratios, none of the transgenic mice had the transgene integrated into the human AAVS1 site.

During the course of this study we have analysed integration sites and expression levels of two different ß-globin gene constructs. The first construct contained LCR core elements HS2 and HS3, the ß-globin gene, and the ß-globin gene 3' enhancer, flanked by insulator elements from the chicken ß-globin gene locus (cHS4). The second construct differed from the first one in that we included the HS2/HS3 flanking sequences. Both of these constructs expressed the ß-globin gene from different positions in the murine genome. However, the construct containing the HS2/HS3 flanking sequence consistently revealed higher ß-globin expression levels, even when integrated in or close to a centromere. This suggests that the HS2/3 flanking region facilitates the activation by LCR core elements.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
We began our studies by generating and analysing transgenic mice harbouring an expression construct of the human ß-globin gene that is small enough to be packaged into recombinant AAV (rAAV, Fig. 1A). This construct contained the core sequences of LCR HS sites 2 and 3, the human ß-globin gene, and the ß-globin 3' enhancer. These elements were flanked on either site with a single copy of an insulator sequence derived from the chicken ß-globin gene locus (cHS4). The rationale for the inclusion of the regulatory elements was the following: HS2 has been shown to have strong enhancer activity when linked to globin or other reporter gene constructs (Ellis et al. 1993) and HS3 has been shown to harbour both enhancer and chromatin opening activity (Ellis et al. 1996). We did not include human LCR element HS4 because results from previous studies suggest that it does not contribute unique activities for ß-globin gene activation (Bungert et al. 1995; Milot et al. 1996; Navas et al. 1998). Chicken HS4 (cHS4) exhibits both enhancer blocking as well as boundary activities (West et al. 2002). Finally, the ß-globin 3' enhancer has been shown to be important for high level ß-globin gene expression in the context of the whole locus in ß-globin yeast artificial chromosome (ß-globin YAC) transgenic mice (Liu et al. 1997). We hypothesized that HS2 and HS3 would open the chromatin regardless of the transgene integration site and that the presence of cHS4 would protect the ß-globin gene expression construct from any negative effect exerted by surrounding chromatin at the site of integration. We have generated and analysed four transgenic lines with this construct. The copy number of the transgene was determined by Southern blotting experiments in which a single copy ß-globin YAC transgene was used as a standard and the murine snrpn gene as an internal control (data not shown). The single copy line contained the entire human ß-globin locus in the context of a YAC (Tanimoto et al. 1999). ß-Globin gene expression in the transgenic lines was analysed by semiquantitative RT-PCR using pairs of primers specific for human ß- and murine {alpha}-globin cDNAs (Bungert et al. 1995). ß-Globin gene expression (ß/{alpha}-globin) was calculated as per cent expression of that of the single copy ß-globin YAC transgenic line (set at 100%). The data are shown in Fig. 1(B) and summarized in panel C. ß-Globin gene expression is presented as expression per copy or total. The data show that expression per copy is low in these transgenic mice, demonstrating that the combination of regulatory elements present in the 432ß4 expression construct is not sufficient to confer high-level ß-globin gene expression. The fact that all of the lines do express the ß-globin gene indicates that the construct is likely protected from position-effects as a result of the presence of the cHS4 insulator elements.



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  		Figure 1  Analysis of a ß-globin expression construct containing LCR core elements HS2 and HS3 as well as the chicken HS4 insulator in transgenic mice. (A) Structure of the ß-globin expression construct 432ß4. The plasmid contained the chicken HS4 insulator sequence, the LCR core elements HS2 and HS3, the ß-globin gene and promoter region and the ß-globin 3' enhancer as indicated. The plasmid was linearized with AatII and injected into pronuclei of fertilized mouse oocytes to generate transgenic lines. Transgenic offspring were mated to generate heterozygous lines. (B) Expression of the human ß-globin gene in the four transgenic lines containing the 432ß4 expression construct (432ß4A to 432ß4D) as well as in a transgenic line containing a single copy of a ß-globin locus YAC (WT). RNA was isolated from anaemic spleen, reverse transcribed and analysed by PCR using primers specific for the mouse (m) {alpha}- and human (h) ß-globin gene as indicated (shown are the signals for 14, 16, 18 and 20 PCR cycles). (C) Summary of expression levels and copy numbers in the individual transgenic lines shown in (B). RT-PCR signals were quantified by phosphor imaging using a storm scanner and imagequant software. Expression levels were calculated based on expression of the mouse {alpha}-globin gene and presented as percentage with expression levels in the human ß-globin YAC transgenic line set as 100%. Copy numbers were determined by Southern blotting experiments with the snrp N gene serving as an internal control and the wild-type ß-globin YAC representing a single copy transgene.

 
Because the inclusion of additional regulatory elements would render the transgene too large for packaging into AAV, we decided to pursue an alternative strategy that, if successful, would allow us to integrate the transgene into a defined site in the mouse genome and which would not be limited by the size of the DNA construct. Wild-type AAV integrates its DNA into a specific site on human chromosome 19, called AAVS1 (Kotin et al. 1992). Although the mechanism of integration is not entirely clear, the process requires cis-acting elements present in the viral genome as well as the function of the AAV encoded rep protein. The AAVS1 sequence was ligated as a 3.6-kb fragment into the EcoRI/KpnI restriction sites of the vector pBS246 leaving a single loxP site at the 3' end of the AAVS1 site. The presence of a single loxP site would allow us to eventually reduce the copy-number by Cre mediated recombination (Garrick et al. 1998). The construct was linearized with EcoRI and used to generate transgenic mice. We generated two transgenic lines, however, only one of these lines continued to transmit the AAVS1 sequence. Southern blotting experiments showed that this line contained the AAVS1 plasmid integrated in 5 tandem copies (data not shown). We mapped the position of the transgene by inverse PCR and DNA FISH analysis. The AAVS1 transgene integrated within the intron of a gene predicted to encode a metalloprotease and located near the telomere of chromosome 15 (Fig. 2).



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  		Figure 2  Generation of human AAVS1 transgenic mice. A 3.6-kb restriction fragment containing the human AAVS1 integration site was cloned into pBS246. The EcoRI linearized plasmid was injected into the pronuclei of fertilized oocytes to generate transgenic mice. Transgenic offspring was mated to generate transgenic S1 founders. (A) Location of the human S1 integration site in the mouse genome. Inverse PCR was used to obtain sequence information from the site of transgene integration. This sequence was blasted against the mouse genomic database (http://www.ensembl.org). A perfect match to the sequence is located close to the telomeric end of chromosome 15. (B) Confirmation of the integration site by DNA FISH. Metaphase spreads of spleen cells from transgenic mice were hybridized using a fluorescent probe specific for the human AAVS1 site (red) and a chromosome paint specific for chromosome 15 (green). DNA was stained with DAPI (blue) and chromosomes were visualized by fluorescence microscopy.

 
We next generated a larger ß-globin expression construct that is similar to the one described in Fig. 1, but contained in addition the flanking region of the HS2 and HS3 core enhancers (43f2ß4, Fig. 3A). Previous work has shown that inclusion of DNA flanking the core regions allows the LCR HS sites to synergistically activate globin gene expression (Molete et al. 2001). The final construct is about 11 kb in size and contains DNA sequence elements derived from the AAV genome, the 5' inverted terminal repeat (ITR) and the p5 promoter region. Both of these sequences have been implicated in the integration of wild-type AAV into the S1 site (Philpott et al. 2002). These elements were placed outside of the 5' cHS4 sequence. The supercoiled plasmid DNA was incubated on ice with recombinant rep68 and the mixture injected into the pronuclei of fertilized oocytes homozygous for the AAVS1 integration site. In vitro chromatin immunoprecipitation (ChIP) experiments have shown that the rep protein binds to the plasmid DNA under these conditions (data not shown). The offspring was first analysed by PCR for the presence of the ß-globin transgene. We generated four transgenic lines with the second ß-globin gene construct (43f2ß4); the first line did not transmit the transgene. The three remaining lines were bred with AAVS1 transgenic mice to generate mice heterozygous for the ß-globin expression construct. Determination of copy number and integrity, as well as expression analysis, was performed as described for the first construct. We also analysed the integration patterns of these mice using DNA FISH. The expression analysis in these mice is summarized in Fig. 3(C). All three lines expressed the ß-globin gene at higher levels than those harbouring the smaller expression construct, supporting previous findings that the flanking sequences allow the HS core sites to synergistically activate ß-globin gene expression (Molete et al. 2001).



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  		Figure 3  Expression analysis of a ß-globin construct containing insulator sequences as well as LCR elements HS2 and -3 with their flanking DNA in transgenic mice. (A) Structure of the 43f2ß4 plasmid. The expression construct contained the 5' ITR and p5 promoter region from AAV, chicken HS4, LCR elements HS2 and HS3 plus flanking DNA, and the ß-globin gene plus 3' enhancer as indicated. (B) Integration pattern of 43f2ß4 transgenic lines (43f2ß4 A and C) analysed by DNA FISH. (C) Summary of human globin gene expression and copy number in transgenic mice harbouring the 43f2ß4 construct (43f2ß4 A to C) or a single copy human ß-globin YAC (WT). Expression of the ß-globin gene and the number of transgene copies was determined as described in Fig. 1(B,C). Expression levels are presented as percentage expression per WT (set at 100%).

 
We next used DNA FISH to determine the site of integration of these constructs in the mouse genome. The results, shown in Figs 3 and 4, demonstrate that the human ß-globin gene construct did not integrate into the transgenic human AAVS1 site on chromosome 15. Recently, a mouse orthologue of the human AAV S1 site has been identified. This sequence is located in the peri-centromeric region of chromosome 7. Because the 43f2ß4B transgene integrated into or close to a murine centromere (Fig. 4A), we examined whether the murine orthologue of the human AAVS1 site has been targeted. However, Southern blotting experiments and DNA FISH with a chromosome 7 paint revealed that it did not integrate into the mouse S1 site (data not shown).



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  		Figure 4  Analysis of the integration site, ß-globin gene expression and DNase I HS sites in transgenic line 43f2ß4B. (A) Analysis of the 43f2ß4B integration site by DNA FISH. Metaphase spreads of spleen cells from transgenic mice were hybridized to fluorescent probes specific for the plasmid 43f2ß4 (red) and chromosome 15 (green), stained with DAPI and visualized by fluorescence microscopy. The insert is a magnification of the region containing the 43f2ß4 integration site. (B) Analysis of DNase I HS sites in the 43f2ß4 transgene. Spleen nuclei of anaemic transgenic mice were digested with increasing concentrations of DNase I. The genomic DNA, isolated from these samples, was digested with EcoRI, size-fractionated by gel-electrophoresis and transferred to a nylon membrane. The DNA was hybridized to a radioactive probe corresponding to a region just 5' of the downstream EcoRI site as indicated. The arrows indicate the position of HS sites associated with LCR element HS2 and the ß-globin promoter. Lane M represents a 1-kb ladder. (C) Analysis of ß-globin gene expression by RT-PCR in 43f2ß4B and ß-globin YAC (WT) transgenic mice. Expression of the human ß- and mouse {alpha}-globin genes was analysed as described in Fig. 1(B). Shown are the signals for 14, 16 and 18 PCR cycles.

 
The line 43f2ß4B expressed the human ß-globin gene at 40% of levels found in a single copy ß-globin YAC transgenic mouse (Fig. 3C). This was the highest level of expression per copy in all of the transgenic lines analysed in this study. The FISH analysis revealed that the transgene integrated into a region located at the centromeric end of a mouse chromosome as indicated by the intense DAPI staining (Fig. 4A). The expression analysis as well as the mapping of DNase I hypersensitive sites demonstrates that the transgene is open and active in this location (Fig. 4B,C).


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Haemoglobinopathies are among the most common inherited diseases in the human population (Higgs et al. 2001). Because of problems associated with current treatments, alternative therapies are highly sought after. Recent advances in gene therapy experiments are promising. For example, investigators have successfully used lentiviral vectors to deliver therapeutic ß-globin expressing constructs into haematopoietic cells of mice carrying mutations in the globin locus (May et al. 2000; Pawliuk et al. 2001). However, the use of lentiviral vectors may not be without problems because the viral DNA is integrated more or less randomly into the genome (Marshall 2002). Adeno-associated virus is considered to be safe because it does not cause strong immune reactions and the DNA of recombinant viruses used in gene therapy experiments often remain episomal (Flotte 2004), although some data suggest that AAV serotype 2 integrates preferentially into transcriptionally active regions in the nucleus (Nakai et al. 2003). The disadvantage of AAV is its low packaging capacity. This is a particular problem for globin gene therapy because it appears that a complex set of regulatory DNA elements is required for expressing therapeutic levels of the globin gene in transgenic assays. An alternative strategy for gene therapy is the development of systems in which the therapeutic DNA can be targeted to a specific and neutral site in the genome.

The attempt to integrate the ß-globin construct into the transgenic human AAV S1 site was unsuccessful. The reason for this is unclear, but it may be related to suboptimal protein or DNA concentrations. Previous work has shown that the components used in our studies are sufficient for AAV to integrate into the S1 site (Philpott et al. 2002). However, the normal route of integration is via viral infection. So it is possible that a critical step is bypassed in the injection procedure. Another possibility is that the transgenic AAVS1 site, which is located within a telomeric region of chromosome 15, is not accessible for recombination in the pronucleus. However, we believe this to be unlikely because transgenic constructs can integrate almost anywhere in the mouse genome, which is the basis of problems associated with position effects. We are continuing our effort to investigate conditions for rep-mediated integration into the transgenic AAVS1 site.

We began our studies by generating a small construct for ß-globin gene expression in transgenic mice. This construct contained the core regions of LCR elements HS2 and HS3, as well as insulator sequences derived from the chicken ß-globin gene locus (cHS4) and the human ß-globin gene plus 3' enhancer. The data show that inclusion of these elements is not sufficient to confer high-level ß-globin gene expression. Low human ß-globin gene transcription could be a result of the fact that the regulatory elements fail to independently establish an active chromatin domain permissible for transcription. The second construct containing the HS2 and HS3 flanking DNA led to higher expression levels in all the transgenes analysed, supporting previous conclusions that the cores function better in the presence of flanking regions (Molete et al. 2001). This is in line with the LCR holocomplex model according to which the HS sites interact with each other to activate globin gene expression (Bungert et al. 1995; Wijgerde et al. 1995). Recent conformational studies suggest that the LCR HS sites are in close proximity in erythroid cells and that genes that are expressed at a specific developmental stage contact the LCR holocomplex (De Laat & Grosveld 2004). Formation of the LCR holocomplex and its association with other HS sites in the globin locus, a complex termed the ‘chromatin hub’ (De Laat & Grosveld 2004), could establish an architecture that is resistant to negative influences exerted by neighbouring chromatin at the site of transgene integration. In this respect, it is possible that the larger construct is able to establish such an architecture or microdomain, whereas the smaller one, because of the absence of the HS core flanking DNA, is unable to do so. Alternatively, the HS 2/3 core flanking region may harbour additional regulatory elements that cooperate with the core HS sites to enhance globin gene transcription (Molete et al. 2001). Indeed, there are evolutionary conserved sequence elements in the HS2/HS3 flanking region that could bind regulatory proteins (Hardison et al. 1997). The presence of the AAV ITR/p5 promoter elements could theoretically influence gene expression. However, we think this is unlikely because these elements were placed outside the cHS4 flanked expression cassette. cHS4 has been shown to exhibit strong enhancer blocking activity (Chung et al. 1997).

The highest level of ß-globin gene expression was observed in a transgenic line in which the construct integrated into a centromeric region. Centromeric or peri-centromeric regions are usually incompatible with transcription, although it has been reported that genes can be expressed within functional centromeres (Saffery et al. 2003). We do not yet know whether the human ß-globin construct integrated into heterochromatic centromeric repeats, but the DNA FISH result shows that the transgene is located at the very tip of the chromosome in a region of intense DAPI staining. The data thus demonstrate that HS2 and HS3, in combination with insulator sequences, are able to confer high-level expression to the ß-globin gene in a centromeric location. It should also be noted that this mouse line carries the lowest number of transgenic copies among all the transgenes analysed here. Copy number itself can influence the expression levels of transgenes. Previous work has shown that a high number of copies can repress transgene expression, possibly because of the fact that highly repetitive DNA tends to fold into a heterochromatic structure (Garrick et al. 1998).

In summary, our data demonstrate that the activity of LCR HS sites is enhanced in the presence of their flanking DNA in transgenic mice. The combination of specific LCR HS sites and boundary elements can provide high-level expression to a ß-globin transgene even when integrated in a centromeric location.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Transgene construction

Components of the chicken and human globin locus were combined to generate the plasmid 432ß4. Briefly, the core region from human HS3 was isolated as an XbaI–XhoI fragment from the plasmid HS434 (Bungert et al. 1995). The HS2 core was generated by PCR from a YAC containing the entire human ß-globin locus as a template (YACA201F4.3; Bungert et al. 1995) using the following primers: HS2US 5'-ACCTCGAGCCCTCTATCCCTTCCAGCATCC-3'; HS2DS, 5'-ACGATTCGAATATCACATTCTGTCTCA-3'; XhoI and EcoRI sites were included 5'-and 3', respectively, to facilitate cloning. The fragments were cloned between the XbaI and EcoRI sites of the plasmid pGEM7 to create pGEM7HS32cores. HS4 from the chicken globin locus was PCR amplified from chicken genomic DNA using primers that introduced AatII and SphI sites 5' and 3' to the 250-bp core element (Chung et al. 1997): 5' HS4US, 5'-ACGACGTCGAGCTCAGGGGACAGCCCCCCC-3'; 5' HS4DS, 5'-GTGGACCCCCTATGCCCCTTTTGCATGCAC-3'. The resulting fragment was cloned into pGEM7HS32cores using AatII and SphI sites. From this plasmid, a SacI-KpnI fragment containing all three core regions (chicken-HS4, Human-HS3 and -2 cores) was isolated and cloned into pUC19 to create pUC432cores. Into this plasmid the human ß-globin gene/3' enhancer was cloned as a 4.6-kb KpnI–XbaI fragment isolated from the plasmid ßA/X (Leach et al. 2001) using KpnI and XbaI sites. Finally the 3' chicken HS4 was PCR generated to contain 5'- and 3'-SalI sites: 3'HS4US, 5'-ATATGTCGACCTCACGGGGACAGCC-3'; 3'HS4DS 5'-CCCGGTCGACCCCCGTATCCCCCCA-3'. The resulting fragment was cloned into the SalI site of pUC432cores to create the plasmid 432ß4pUC. The plasmid containing the ß-globin integration construct (p43f2ß4) was constructed using the pNEB193 plasmid (New England Biolabs) as the backbone. A 500-bp EcoRI-PstI fragment containing the AAV2 5' ITR and p5 promoter was blunt ended on the 3' end and cloned into the EcoRI and SmaI sites of pNEB193. A linker containing PacI, SpeI, NsiI, ApaI and XbaI restriction enzyme sites were then cloned into the PacI and XbaI sites. The 246-bp 3' cHS4 fragment was PCR amplified using the 3' HS4 primers described above and ligated into the SalI site of the vector. Next a 3.8-kb NsiI–XbaI fragment containing the human ß-globin gene, including the promoter and 3' enhancer elements, was cloned into the respective sites in the vector. A 6.9-kb NsiI fragment containing the human ß-globin LCR from HS3 through HS2 was ligated into the vector and clones with the correct orientation were identified by restriction enzyme analysis. The 5' HS4 fragment was PCR amplified using the following primers: HS4 US, 5'-CCTTAATTAACTCACGGGGACAGCC-3', HS4DS 5'-CTAGTCTAGACCCCGTATCCCCCA-3', introducing PacI and XbaI sites at the 5'-and 3' ends, respectively. This fragment was ligated into the PacI and SpeI sites of the vector. This cloning step removed 742 bp of the 5' end of the HS3 flank HS2 LCR fragment previously ligated into this vector. The complete p43f2ß4 plasmid was purified using Qiagen's maxiprep kit. Both plasmids were sequenced to verify the integrity of the regulatory elements.

The 3.6-kb EcoRI-KpnI fragment containing the human AAVS1 site was ligated into pBS246 (Invitrogen).

Transgenic mouse production

We used FvBN{gamma} mice (Jackson Laboratories) to generate all transgenic lines. Plasmid p432ß4 was linearized with AatII; pAAVS1 was linearized with EcoRI. The linearized plasmids were purified from agarose gels and re-suspended in injection buffer at a concentration of 2 ng/µL. Transgenic mice were generated as previously described (Bungert et al. 1995). Transgenic founders were first identified by PCR on DNA isolated from tail clips. Copy number and integrity was analysed by Southern blotting. AAVS1 transgenic mice were mated to generate mice homozygous for the transgene. In experiments using the ß-globin integration construct (p43f2ß4) 1–5 ng of the supercoiled plasmid DNA was complexed on ice with a 1 : 5, 1 : 10 or 1 : 15 molar ratio of DNA to purified AAV2 rep68 protein and injected into fertilized oocytes homozygous for the AAVS1 transgene.

DNA isolation, PCR screening, inverse PCR and Southern blot analysis

DNA was isolated from mouse tail and the presence of human AAVS1 or ß-globin locus sequences was first determined by PCR using primers against the human AAVS1 site and the flanking region between human ß-globin HS3 and HS2: AAVS1 US 5'-ATCTGCCCGGCATTTCTGAC-3', AAVS1 DS 5'-CGCAAAATGTCGCAAAACAC-3'. The primers amplifying a region between HS2 and HS3 were published by Leach et al. (2003). DNA from tails that contained AAVS1 and/or human ß-globin sequences were then subjected to Southern blot analysis. Approximately 10 µg of tail DNA was digested with restriction enzymes, size fractionated on 1.2% agarose gels and transferred on to nylon membranes as described (Bungert et al. 1999). The membranes were then probed with DNA fragments corresponding to regions of the ß-globin transgene or of the human AAVS1 site. The ß mid probe is a 917-bp BamHI–EcoRI fragment derived from pßA/X (Leach et al. 2001) and corresponds to the coding region of the human ß-globin gene. The 3' ß-globin probe is a PstI fragment encompassing the ß-globin 3' enhancer and derived from pßA/X. The AAVS1 probe was derived by PCR using the primers described above (AAVS1 US and DS). Copy number of transgenes was determined by hybridizing the nylon membranes with a radioactive probe corresponding to the murine snrp N gene. This probe is a 300-bp EcoRI/SacI fragment derived from the snrp N locus which hybridizes to a 4-kb EcoRI fragment in Southern blotting experiments. This probe was made available to us by Dr Camilynn Brannan (University of Florida).

Inverse PCR was carried out as described by Hartl & Ochman (1996). Briefly, genomic DNA from S1 transgenes was digested with SacI or MspI, ligated and subjected to PCR using the following primers, S1tg DSI: 5'-CACAGCCCCAGGTGGAGAAACT-3', S1tg DSII: 5'-CCCGGGTTGGAGGAAGAAGACT-3', S1tg US: 5'-TTCTCCAGGCAGGTCCCCAA-3'. PCR products were subcloned into the TopoII vector (Invitrogen) for sequencing.

Metaphase preparation and FISH analysis

F1 animals containing the transgene were killed and the spleens were isolated in 2-3 mL sterile phosphate-buffered saline (PBS) for metaphase chromosome preparations (Trask et al. 1991). Cells were isolated from the spleen and pelleted in a total volume of 10 mL PBS at 500 g for 10 min. The cells were then immediately re-suspended in 10 mL 0.075 M KCl (pre-warmed to 37 °C) and incubated at 37 °C for 20 min 2 mL fixative (3 parts methanol : 1 part glacial acetic acid) was immediately added to the cells and the cells were pelleted at 500 g for 10 min. The metaphase cells were washed three times in 10 mL fixative and stored at 4 °C in fixative.

Metaphase cells were placed on microscope slides and allowed to air dry. The slides were aged in an 80 °C incubator for 1 h and then immediately used for FISH. In the dark, 10–15 µL fluorescently labelled probe was placed on each slide, covered with a glass slide, and then placed in a HyBrite (Vysis) apparatus overnight where the slides were denatured at 75 °C for 15 min and hybridized at 37 °C for 16 h. Cover glasses were then removed in the dark, and the slides were washed for 2 min at 75 °C in 0.4 x saline sodium citrate buffer (SSC), 0.3% NP-40, followed immediately by one wash in 2 x SSC, 1% NP-40 for 1 min at room temperature. The slides were allowed to air dry in the dark and counterstained with 10 µL DAPI II solution (Vysis). The slides were visualized by fluorescence microscopy or stored in the dark at 4 °C.

The entire ß-globin transgene (p43f2ß4) or the AAVS1 plasmid was fluorescently labelled using the BioPrime kit (Invitrogen) by substituting rhodamine-tagged dUTP (tetramethylrhodamine-5-2'-deoxy-uridine-5'-triphosphate, Roche) for the biotin-tagged dUTP that comes with the kit. The chromosome paint probes, specific for mouse chromosomes 7 and 15, were obtained from ID Laboratories Inc. and used according to the manufacturer's instruction.

RNA isolation and semiquantitative RT-PCR

F1 animals containing the ß-globin transgene were made anaemic by injecting phenylhydrazine as previously described (Bungert et al. 1995). RNA was extracted from the spleen and cDNA was prepared as described in Bungert et al. (1995). Ten per cent of the RT reaction was used for semiquantitative PCR analysis using primers against the human ß-globin and mouse {alpha}-globin genes using primers published previously (Bungert et al. 1995). We used a new mouse {alpha}-globin downstream primer to span an intron in these experiments: m{alpha} DS, 5'-TCCACACGCAGCTTGTGGGCATGCAG-3'. PCR samples were removed at 14, 16 and 18 cycles and size fractionated on a 10% polyacrylamide gel. The gels were stained with SyBr-green and quantified by phosphorimager analysis using a storm scanner. Human ß-globin transgene expression level was calculated relative to the expression level of the endogenous mouse {alpha}-globin gene.

DNase I hypersensitivity analysis

Cells taken from a spleen of mice made anaemic by phenylhydrazine injection (see RNA isolation section) were washed with PBS, pelleted and subjected to DNase I digestion as described by Kang et al. (2003). DNase I (10 µg)-treated DNA was digested with EcoRI, size fractionated on a 0.8% agarose gel and subjected to Southern blot analysis using the ß-mid probe as described above.


   Acknowledgements
 
We thank Gail Green for technical assistance and our colleagues in the Bungert laboratory for helpful discussions. We thank Drs Nick Muzyczka and Kevin Nash (UF) for providing us with recombinant rep protein. We thank the members of the RC Philips Cytogenetic Laboratory and Ahmad Kahlil (UF) for help with DNA FISH and the Transgenic Core facility (UF) for letting us use the equipment for microinjections. We thank Camilynn Brannan for the snrp N gene plasmid and for helpful suggestions. This work was supported by grants from the NIH (DK058209 and DK52356) and American Heart Association (to JB).


   Footnotes
 
Communicated by: Masayuki M. Yamamoto

{dagger}S. H. L. Kang and P. P. Levings contributed equally to this work. Back

* Correspondence: E-mail: jbungert{at}ufl.edu


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
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Received: 28 June 2004
Accepted: 10 August 2004




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