|
http://www.abbs.info e-mail: [email protected] ISSN
0582-9879
ACTA BIOCHIMICA et
BIOPHYSICA SINICA 2003, 35(10): 947－951
CN 31-1300/Q |
Short Communication
|
Cre-mediated Site-specific
Cassette Exchange in Erythroid Cell
LI Xing-Guo, YAN Hao-Heng, LIU De-Pei*, HAO De-Long, LIANG Chih-Chuan
( National Laboratory of Medical Molecular
Biology,
Abstract Cre-mediated cassette exchange has been developed to perform site-specific chromosomal integration using Cre recombinase. Here, site-specific integration with inverted Lox sites was used to investigate the erythroid cis-acting DNA element in specific chromatin contexts in mouse erythroleukemia cells. Single hygromycin-resistant clones were obtained from the selective semi-solid medium containing hygromycin post-electroporation. PCR and Southern blotting analysis showed single-copy integration of target vector in clones A, B and D. Site-specific cassette exchange was performed in clone A with exchange vector and Cre expression plasmid, followed by gancyclovir selection. Flow cytometry was used for analysis of EGFP gene expression. A 732-bp fragment of human β-globin gene cluster 5′ DNase I hypersensitive site 2(HS2) was exchanged and integrated into clone A in an anti-genomic orientation. The low EGFP expression in clone A-HS may be due to the orientation-dependent gene silencing caused by integration of HS2 in a non-permissive orientation.
Key words chromosomal integration; site-specific recombination; Cre/Lox; globin gene; gene silencing
Many techniques available for stable integration of transgenes in mammalian cells often result in integration at random chromosomal locations of multiple copies of transgenes that express at levels that are difficult to be predicted or reproduced because of position effects. While homologous recombination is the common solution for embryonic stem cells, the efficiency of stable integration of transgenes is largely masked in permanent cell line by massive illegitimate recombination event[1]. An alternative way was to utilize the site-specific recombinases adapted from phages or yeast (Cre or Flp, respectively)[2]. The target site of the Cre recombinase (Lox site) is a 34-bp sequence that consists of two inverted 13-bp Cre-binding sites linked by an 8-bp spacer within which the recombination occurs[3] (Fig.1). Two recombination target sites will not recombine with different central 8-bp-spacer region, whereas recombine efficiently with same spacer region. A chromosomal cassette flanked by two hetero-specific Lox sites can therefore be replaced by another cassette (located on a plasmid) flanked by such mutually incompatible Lox sites through a double reciprocal recombination[4]. Cre-mediated site-specific chromosomal integration in mammalian cells has been previously shown feasible, although with relatively low efficiency[5]. Recent reports have demonstrated that the mutated hetero-specific Lox sites (LoxP 511 and LoxP, for example) are not entirely incompatible, and that an unexpected excision reaction occurred between such Lox sites due to recombination between them[6].
Fig.1 Structure
of a Lox site
The target site of the Cre recombinase is a 34 bp DNA sequence that consists of two inverted 13 bp Cre-binding sites linked by an 8-bp spacer within which recombination occurs.
In the present study, a modified strategy based on inverted Lox sites[L1 (LoxP 511) and 1L (inverted LoxP 511)] was employed to investigate the erythroid cis-acting DNA element, a 732-bp fragment of human β-globin gene cluster 5′ DNase I hypersensitive site 2(HS2), in specific chromatin contexts of mouse erythroleukemia cells (MELc). Intra-chromosomal recombination between two inverted Lox sites would lead to inversion rather than excision, and would therefore maintain the high efficiency of recombination[6] (Fig.2). The positive selection, negative selection and orientation of cassette exchange were discussed.
Fig.2 Cre-mediated
cassette exchange
The classical RMCE event generally goes as the following steps. Firstly, the target vector is randomly integrated into the cellular genome. Both the target sequence and the exchange sequence are flanked by two inverted Lox sites (LoxP 511 and inverted LoxP 511, designated as L1 and 1L respectively). After co-transfection of the exchange vector and Cre expression vector, the target sequence between inverted Lox sites can be readily recombined to the exchange vector containing the same pair of inverted Lox sites.
1 Materials and Methods
1.1 Plasmids
Plasmid
construction was performed according to the conventional techniques[7]. The
target vector p1L-HyTk-L1-β-EGFP-IRES-neo (designated as β-EGFP-neo thereafter)
was constructed in three steps. First, a PCR product of human β-globin promoter
(309 bp, HUMHBB positions 61 921 to 62 230) was inserted into the SmaI site of
plasmid pEGFP-1 (
pL1-HS2-1L was constructed from pL1-HyTk-1L by replacing the HyTk sequence with HS2 (732 bp, HUMHBB positions 8486 to 9218) which was a HindIII/BglII core fragment of 5′ HS2 of human β-globin gene locus control region.
1.2 Cell culture and creation of
hygromycin-resistant clones
MELc were grown
in Dulbecco’s modified
Eagle’s medium
(DMEM) containing 10% fetal bovine serum. Electroporation was performed as
follows: 5×106 MELc in
the mid-to-late logarithmic phase of growth in 400 μL of PBS was mixed with 10 μg of Eco31I(MBI Fermentas,
USA)-linearized β-EGFP-neo, and electroporated at 280 V and 950 μF in a gene pulser (Bio-Rad, USA). The
selective semi-solid medium containing 2% methyl cellulose (
1.3 Cre-mediated site-specific cassette
exchange
Clone A was
co-electroporated with 25 μg pL1-HS2-1L and 15 μg Cre expression vector pBS185 (Life Technologies, USA). Negative
selection with 3.0 g/L gancyclovir (Gcv) (
1.4 Flow cytometry analysis
Flow cytometry
was performed on a Becton Dickinson instrument (FACS Calibur,
2 Results
In target vector the β-globin gene promoter is located upstream of the coding sequence of two reporter genes: EGFP and neo were interposed by internal ribosome entry site (IRES) sequence. The transcriptional direction of reporter genes is opposite to that of selectable marker HyTk gene (Fig.3).
Fig.3 Target
vector pβ-EGFP-neo and exchange vector pL1-HS2-1L
The target vector pβ-EGFP-neo includes the CMV-HyTk gene and can be selected positively by Hyg and negatively by Gcv. The arrow indicates the transcriptional orientation of reporter genes driven by a 309-bp human β-globin gene promoter. The thick line below stands for 309-bp human β-globin gene promoter as the probe used in Southern blotting analysis of XbaI-digested genomic DNA.
PCR and Southern blotting analysis indicated that three hygR clones (clone A, B and D) contained single copy of target vector pβ-EGFP-neo (Fig.4, 5). For each of these three clones, flow cytometry analysis showed no significant differences among subclones both in the percentage and in the mean fluorescence intensity (MFI) of EGFP-expressing cells (data not shown).
Fig.4 PCR
analysis of genomic DNA of four hygR single clones with upstream primer of
human β-globin gene promoter and downstream primer of EGFP gene (PCR product:
779 bp) and specific Neo gene primers (PCR product: 432 bp)
M, 100 bp DNA ladder; N, negative control; P, plasmid pβ-EGFP-neo as the positive control; H, human genomic DNA; Mc, MELc; A, clone A; B, clone B; C, clone C; D, clone D.
Fig.5 Southern blotting analysis of XbaI digested genomic DNA of three hygR single clones with the 309-bp of human β-globin gene promoter as the probe
P, plasmid pβ-EGFP-neo as the positive control; N, negative control; Mc, MELc; A, clone A; B, clone B; D, clone D.
PCR analysis also indicated that the HS2 fragment was exchanged and integrated into gcvR clone A-HS in an anti-genomic orientation (Fig.6). There were no significant differences among the subclones of clone A-HS2 (data not shown) while the mean percentage of EGFP-expressing cells was much lower (2.42%) than that in clone A (35.94%) (Table 1).
Fig.6 PCR
products amplified by primers of β-globin promoter and HS2 core primers
M, 100 bp DNA ladder; N1, negative control (β-globin promoter primers);
P1, plasmid pβ-EGFP-neo (PCR product: 309 bp of human β-globin gene promoter
sequence); A1, clone A; A-HS1, clone A-HS; N2, negative control (HS2 core
primers); P2, plasmid pL1-HS2-1L (PCR product: 247 bp of 5′ HS2 core
sequence); A2, clone A; A-HS2, clone A-HS; N3, negative control (downstream
primer of HS2 & downstream primer of β-globin promoter); A3, clone A;
A-HS3, clone A-HS (PCR product: 748 bp); N4, negative control (upstream primer
of HS2 & downstream primer of β-globin promoter); A4, clone A; A-HS4, clone
A-HS.
Table 1 Comparison
of EGFP expression in MELc, clone A and clone A-HS2 (un-induced and induced by
2% DMSO or 75 μmol/L hemin)
|
|
Percentage of EGFP-expressing
cells (%) |
Mean fluorescence intensity
(MFI) |
|
|
MELc |
3.49 |
18.51 |
|
|
A |
35.94 |
22.34 |
|
|
A-HS2 |
2.42 |
26.66 |
|
|
Induced MELc |
DMSO |
12.72 |
22.84 |
|
Hemin |
1.20 |
20.01 |
|
|
Induced A-HS2 |
DMSO |
9.09 |
27.38 |
|
Hemin |
1.55 |
15.54 |
|
3 Discussion
The Cre-mediated cassette exchange strategy uses site-specific recombinase to integrate single-copy transgene without selective markers into previously tagged sites in mammalian cells[8]. In this study we described a simple method for site-specific integration in erythroid cells based on the use of inverted Lox sites to investigate the erythroid cis-acting DNA element in specific chromatin contexts. PCR and Southern blotting analysis showed single-copy integration of target vector pβ-EGFP-neo in HygR clones A, B and D. A 732-bp fragment of 5′HS2 was exchanged and integrated into clone A in an anti-genomic orientation. The low EGFP expression in clone A-HS may be due to the orientation-dependent gene silencing caused by integration of HS2 in a non-permissive orientation.
The target vector pβ-EGFP-neo includes the CMV-HyTk gene and can be selected positively by Hyg and negatively by Gcv. Gcv, a non-toxic pro-drug, is converted to a phosphorylated active analog and is incorporated into the DNA of replicating eukaryotic cells, causing death of dividing cells, upon expression of a viral suicide gene encoding thymidine kinase (Tk). It has been widely used in molecular biology for selection against random recombination events when homologous recombination of a gene of interest is required. There have been several reports describing the strong sensitivity of HyTk-expressing cells to Gcv (>99% cell death in 48 h in 10 μmol/L Gcv)[9]. Our results indicated that hygR clones A, B, and D showed differential sensitivity to Gcv, with lethal concentration of Gcv in 48 h at 10 μmol/L, 20 μmol/L, and 30 μmol/L respectively (data not shown). Due to the limited number of Gcv clones in this study, we still cannot rule out the possible relationship between Gcv sensitivity and exchange efficiency at different chromosomal sites of integration[10].
In contrast to recessive markers for negative selection, many dominant markers for positive selection were isolated from bacteria as autonomously replicating plasmid DNA molecules carrying genes for resistance to antibiotics. G418 and Hyg are two widely used selective reagents produced by streptomycetes[11]. As this kind of antibiotics exhibit broad specificity in prokaryotes and in eukaryotes as diverse as yeast and mammals, it makes positive selection of stably transfected suspension cell clones from semi-solid medium containing appropriate antibiotics an especially simple and efficient method (for example, selective semi-solid medium containing 2% methyl cellulose in the selection of hygR clones in this study). As for negative selection, however, semi-solid medium selection didn’t show any obvious advantages over other routine clone expansion methods. In general, stably transfected cells selected for either G418 or Hyg resistance will maintain the plasmid sequences in the absence of drug selection for as many as 50－75 cell doublings[12]. Prior to Cre-mediated cassette exchange, however, it is necessary to select HyTk-expressing cell clones in selective medium for 2－3 weeks to ensure that all cells express the HyTk gene[13].
5'-HS2 is a well-characterized erythroid enhancer. A 732-bp HindIII/BglII core fragment showed enhancer activity that was erythroid-specific and developmental-stage nonspecific[14]. DNA fragments containing HS2 will confer position-independent, high level expression on globin genes in transgenic mice. When tested in cell transfection assays, HS2 greatly enhances expression of β-like globin genes in an erythroid-specific manner, both in transient expression from un-integrated constructs and after stable integration into a chromosome[15]. Transgenic studies suggested that HS2 linked to the β-globin gene in the genomic orientation can activate high-level human β-globin gene expression in the erythroid tissues of transgenic mice, whereas HS2 in the anti-genomic orientation appears to predispose the construct to significant rearrangements[16]. A recent report demonstrated orientation-specific silencing of 5′-HS 234 fragment of human β-globin locus control region occurred in the two distinct orientations at the same integrated locus in MELc[9].
Due to the nature of inverted Lox sites, the cassette exchange will occur in an unexpected manner: in one orientation in half of the clones and the reverse one in the other half. In our study, PCR analysis showed the anti-genomic integration of HS2 fragment in clone A-HS2. Similarly, clone A-HS2 was also not sensitive to DMSO or hemin induction, in contrast to the regular induction patterns of these reagents (Table 1). The reporter gene suppression in A-HS2 may be due to the orientation-specific gene silencing caused by non-permissive integration. Further study on the reversion from non-permissive orientation to permissive orientation of HS2 in A-HS may provide clue to understanding the mechanisms underlying orientation-dependent gene silencing and effect of integration site on gene expression and regulation. The cell clones A, B and D contain inverted Lox sites in the artificial genomic loci and may provide the source of recipient cell strains for further steps toward systematic screening and functional elucidation of cis-acting elements in the specific chromatin contexts[17].
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_________________________________________________
Received:
This work was supported by the grants from the National Natural Science Foundation of China (No. 30393110) and the Science Foundation for Chinese Outstanding Youth (No. 3952006)
*Corresponding author: Tel, 86-10-85116436-8514; Fax, 86-10-65133086; e-mail, [email protected]
Updated at: 2003-10-09
红系细胞中Cre重组酶介导的位点特异性片段交换
李兴国 严皓珩 刘德培* 郝德龙 梁植权
( 中国医学科学院基础医学研究所， 中国协和医科大学基础医学院医学分子生物学国家重点实验室， 北京 100005 )
摘要 re介导的片段交换技术利用重组酶Cre的位点特异性重组特性， 在基因组的特定位点进行靶片段与目的片段的交换。 运用互为反向的Lox位点， 在鼠红白血病MEL细胞中进行靶载体的整合和交换载体的交换， 探讨在特定的染色质环境下红系特异性顺式作用元件的功能。 电穿孔转染MEL细胞后从含有潮霉素（hygromycin）的选择性半固体培养基中挑取MEL细胞单克隆， 通过PCR和Southern杂交鉴定整合完整性和拷贝数， 获得三种整合有靶载体p1L-HyTk-L1-β-EGFP-neo的细胞株A， B和D。 交换载体pL1-HS2-1L（含有732-bp的人β-珠蛋白基因簇5′ DNase I 高敏位点2核心片段）和Cre表达载体pBS185共转染细胞株A， 9-(1,3-二羟-2丙氧甲基)鸟嘌呤(gancyclovir)负筛选后挑取单细胞克隆A-HS。 PCR检测显示HS2片段以反方向进行了交换。 流式细胞仪分析显示平均的荧光细胞百分比（2.42 %）低于未交换的细胞株A （35.94 %）。 A-HS中EGFP的低表达可能是处于非容许方向的HS2片段出现方向依赖性基因沉默所致。
关键词 色体整合； 位点特异性重组； Cre/Lox； 珠蛋白基因； 基因沉默