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An effective alternate cloning strategy for unstable mouse genomic sequences
BBRC Biochemical and Biophysical Research Communications 330 (2005) 641–644 www.elsevier.com/locate/ybbrc
An effective alternate cloning strategy for unstable mouse genomic sequences Michael S. Lan *, Michelle Muguira The Research Institute for Children, ChildrenÕs Hospital, Departments of Pediatrics and Genetics, Louisiana State University Health Sciences Center, New Orleans, LA 70118, USA Received 25 February 2005 Available online 19 March 2005
Abstract Unstable mammalian genomic sequences frequently underwent spontaneous rearrangement during the bacterial cloning process. When the flanking sequences of an INSM1 gene comprised of 3.0 and 4.5 kb were subcloned into a targeting vector for a gene deletion study, both the genomic sequences underwent spontaneous rearrangement. Neither the usage of recombinase-free Escherichia coli competent cells nor lowering the culture incubation temperature averted the recombination events. Co-transformation of a methyltransferase vector, pAIT2, with the targeting vector had little effect in preventing recombination through methylation of the plasmid DNA. Here, we show that a single-copy cloning technique is effective to clone the unstable mouse genomic DNA into the targeting vector. Ó 2005 Elsevier Inc. All rights reserved. Keywords: INSM1; Recombination; Unstable genomic sequences; pBAC/oriV vector; Copy control cloning
The insulinoma-associated 1 (INSM1/IA-1) gene encodes a 510 amino acid zinc-finger transcriptional repressor originally isolated from a human insulinoma subtraction library [1,2]. Tissue-specific expression patterns revealed that the INSM1 gene is developmentally regulated in the pancreas and nervous system, as well as in tumors of neuroendocrine origin [3–5]. The INSM1 gene is located on human chromosome 20p11.2 and on mouse chromosome 2. Both were identified as intronless genes [4,6]. The flanking sequences of the INSM1 gene were first PCR amplified from the genomic DNA of the mouse strain 129/SVJ. The genomic organization of the INSM1 gene and restriction enzyme mapping were determined by sequence analysis. In order to elucidate the biological function of the INSM1 gene, a conditional targeting construct was designed for a gene knockout experiment. A pair of loxP *
Corresponding author. Fax: +1 504 896 2722. E-mail address: [email protected] (M.S. Lan).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.022
sites was designed to flank the partial INSM1 promoter sequence and the entire coding sequence. The 5 0 -arm (3.0 kb) and 3 0 -arm (4.5 kb) of the INSM1 genomic sequences were subsequently cloned into the flanking regions of the INSM1 gene for homologous recombination. In this study, both the 5 0 - and 3 0 -arm genomic sequences experienced spontaneous rearrangement during the bacterial cloning processes. After testing several cloning strategies, we succeeded in using a copy-control vector that limits the rearrangement events for these unstable genomic DNA sequences. This cloning technique could be of particular use when dealing with unstable genomic sequences during the construction of targeting vectors for gene knockout experiments.
Materials and methods FRT-loxP targeting vector construction. The FRT-loxP vector was kindly provided by Dr. C. Wright (Vanderbilt University, Nashville,
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M.S. Lan, M. Muguira / Biochemical and Biophysical Research Communications 330 (2005) 641–644
TN). A HindIII fragment (3.5 kb) consisting of a partial promoter and the entire INSM1 coding sequence was subcloned into the FRT-loxP vector. The orientation of the insert was determined by sequence analysis. The 5 0 -arm (3.0 kb) and the 3 0 -arm (4.5 kb) of the INSM1 gene were PCR amplified with appropriate flanking enzyme cloning sites from 129/SVJ genomic DNA, using a high fidelity DNA polymerase (Invitrogen). Subsequent cloning processes were carried out using maximal efficient electrocompetent cells (XL10-Gold). Co-transformation of pAIT2 with the targeting vector for in vivo methylation. It has been reported that CpG methylation modifies the genetic stability of cloned repeat sequences [7]. In order to test the hypothesis that the CpG-rich region of the INSM1 gene may contribute to DNA instability, we employed a pAIT2 in vivo methylation system. Both the INSM1 targeting and pAIT2 vectors were co-transformed into Escherichia coli competent cells, and selected for both ampicillin and kanamycin resistant clones. Copy control single-copy cloning strategy. The very low copy bacterial artificial chromosome (BAC) vector is frequently used for its advantage of maintaining a high stability of inserted clones. A copy control PCR cloning kit (Epicentre) was used in our study. The FRTloxP-INSM1 fragment was digested with SalI/NotI, blunt-ended, and subcloned into the PCR cloning vector using a blue-white selection system. An electrocompetent TransforMax EPI300 E. coli that can be induced to a high-copy number within 5 h was used for this vector. The electroporation condition was modified to achieve high transformation efficiency since the vector size was over 15 kb. A conventional colony hybridization method was employed to select positive clones using the labeled insert as a probe.
Results and discussion The partial promoter and the entire coding sequence were HindIII cloned into the FRT-loxP targeting vector. However, the subsequent cloning of either the 5 0 -arm (3.0 kb) or 3 0 -arm (4.5 kb) sequences into either end of the targeting construct failed to generate a vector of the correct size (Fig. 1). The starting FRT-loxP vector, with the loxP sites flanking the promoter and the INSM1 coding sequence, represented a size of 11 kb. However, after the ligation of either the 5 0 -arm or 3 0 arm into the vector, the vector size was rearranged into 63.0 kb. Similar experimental results were obtained in many attempts of this simple cloning process. Therefore, we concluded that an unexpected recombination event must be occurring spontaneously during the cloning process. Analysis of the INSM1 flanking sequence and INSM1-3 0 untranslated sequence revealed tandem copies of CAn (n = 38) and CTn (n = 28) dinucleotide repeats, and the highly GC-rich sequences in the promoter region of the INSM1 gene. It is speculated that these repeat sequences could cause the recombination occurrence. In an effort to eliminate the recombination, two E. coli strains were selected, such as ElectroMAX Stbl4 (Invitrogen) and SURE2 (Stratagene), that reportedly improve the cloning capacity of repeat containing insert sequences [8]. The culture temperature was lowered to 25 °C, which should also help to prevent recombination. However, after screening over a hundred clones neither change showed any advantage
Fig. 1. Molecular cloning of the 5 0 -arm and 3 0 -arm sequences of the INSM1 gene caused rearrangement. The FRT-loxP-INSM1 vector (11 kb) was digested with SalI and ClaI, and ligated with the 5 0 -arm (3 kb, SalI/ClaI) overnight. The ligation mixture was transformed into XL10-Gold competent cells. Over a hundred colonies from at least five experiments were analyzed by SalI and ClaI digestion. A 2.8 kb band was present in all of the clones selected for analysis. Similarly, the FRT-loxP-INSM1 vector was digested with BamHI, treated with calf intestine phosphatase, and then ligated with the 3 0 -arm (4.5 kb, BamHI) overnight. The ligation mixture was transformed into XL10Gold competent cells. Over a hundred colonies from at lease five experiments were analyzed by BamHI digestion. Two bands, 3 and 2.2 kb, were present in the majority of the clones selected for analysis. The same results were observed in different experiments and are likely due to unstable genomic sequence rearrangement in the bacteria.
over the regular XL10-Gold competent cells that we normally use. We further evaluated whether CpG methylation could improve the genetic stability of the cloned repeat sequences [7]. The transformation was performed with the addition of an E. coli-based in vivo methylation system-pAIT2 (KanR). Two selection systems (AmpR and KanR) allowed the selective growth of cells containing both plasmids upon co-transformation. Since genomic stability is not solely dependent upon the presence of repeated sequences, co-transformation of the pAIT2 vector with the INSM1 genomic sequences did not inhibit the genomic sequence recombination (data not shown). An alternate strategy developed by Shizuya et al. [9] was based on the concept of using very low copy bacterial artificial chromosome vectors (BACs) for unstable genomic sequences. The disadvantage of a low copy number BAC vector was overcome by the development of a novel class of inducible single-copy/high-copy pBAC/oriV vectors [10]. This single-copy cloning technique could both greatly reduce the probability of recombination during the cloning process, and compensate for a low copy number by the conditional induction
M.S. Lan, M. Muguira / Biochemical and Biophysical Research Communications 330 (2005) 641–644
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Fig. 2. Construction of the INSM1 targeting vector into the copy-control pCC1 vector. The partial promoter and the entire INSM1 coding sequence (HindIII fragment, 3.5 kb) was cloned into a pFRT-loxP vector, named FRT-loxP-INSM1. The FRT-loxP-INSM1 fragment was digested with SalI and NotI, blunt-ended, and cloned into the copy-control pCC1 vector. The 5 0 -arm of the INSM1 gene (3 kb) was PCR amplified to include a ClaI site at both ends for pCC1 vector cloning. Subsequently, the 3 0 -arm of the INSM1 gene (4.5 kb) was PCR amplified with BamHI sites present at both ends for cloning into the FRT-loxP-INSM1 targeting vector. The electroporation voltage was modified by using a 0.1 cm cuvette with 1.5 kV setting. The orientation of each insert was determined by sequence analysis. The entire targeting sequence (15.5 kb) was digested with NotI sites from the copy-control pCC1 vector for ES cell transfection studies.
of the high-copy number through the trf A gene product supplied by the TransforMax EPI300 E. coli (Epicentre). This strategy was tested in our system by first blunt-end cloning the FRT-loxP-INSM1 (SalI/NotI fragment) into the pCC1 blunt-end cloning vector, using the blue-white selection method. The copy control clones can be induced to produce up to 200 plasmid copies per cell in 5 h [10]. This level of induction provides enough plasmid DNA for sequence analysis. The positive clones were subsequently subjected to the subcloning of the 5 0 -arm (ClaI site) followed by the 3 0 -arm (BamHI site) sequences into the copy-control pCC1 vector (Fig. 2). The cloning efficiency decreased drastically as the vector size increased to over 15 kb, and the electroporation condition had to be adjusted with a higher voltage setting. Positive clones were selected with a colony hybridization method. The cloning process was tedious due to the selection of unique restriction enzyme sites and the blunt-end ligation approach, but successfully eliminated the recombination event. Completion of the human genome project provides immense information for human genetics. The next task is to elucidate the biological function of each individual gene. Genetic instability always presents difficulties in laboratory manipulation of unstable genomic sequences. It is reported here that the flanking arm sequences of an intronless gene, INSM1, caused recombination, making them unclonable in bacteria. After testing various options, we showed that the commercially available copy-
control PCR cloning kit (Epicentre) provided an effective alternative for unstable genomic DNA cloning, which is essential for pursuing our gene targeting experiments.
Acknowledgments We thank Drs. S.H. Pincus, M.B. Breslin, M. Ferris, and D.S. Fox, for critical reading of the manuscript, Dr. B. Jack (New England BioLabs, Beverly, MA) for the pAIT2 vector, and Dr. C. Wright (Vanderbilt University, Nashville, TN) for the FRT-loxP vector. This work was supported by funds from the Research Institute for Children, ChildrenÕs Hospital, and a grant from NIDDK, National Institutes of Health (NIH) DK61436 (to M.S.L.).
References [1] Y. Goto, M.G. DeSilva, A. Toscani, B.S. Prabhakar, A.L. Notkins, M.S. Lan, A novel human insulinoma-associated cDNA, IA-1, encodes a protein with zinc-finger DNA-binding motifs, J. Biol. Chem. 267 (1992) 15252–15257. [2] M.B. Breslin, M. Zhu, A.L. Notkins, M.S. Lan, Neuroendocrine differentiation factor, IA-1, is a transcriptional repressor and contains a specific DNA-binding domain: identification of consensus IA-1 binding sequence, Nucleic Acids Res. 30 (2002) 1038– 1045.
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M.S. Lan, M. Muguira / Biochemical and Biophysical Research Communications 330 (2005) 641–644
[3] M.B. Breslin, M. Zhu, M.S. Lan, NeuroD1/E47 regulates the Ebox element of a novel zinc-finger transcription factor, IA-1, in developing nervous system, J. Biol. Chem. 278 (2003) 38991–38997. [4] J.P. Xie, T. Cai, H. Zhang, M.S. Lan, A.L. Notkins, The zincfinger transcription factor INSM1 is expressed during embryo development and interacts with the Cbl-associated protein, Genomics 80 (2002) 54–61. [5] M. Zhu, M.B. Breslin, M.S. Lan, Expression of a novel zincfinger cDNA, IA-1, is associated with rat AR42J cells differentiation into insulin-positive cells, Pancreas 24 (2002) 139–145. [6] M.S. Lan, Q. Li, J. Lu, W.S. Modi, A.L. Notkins, Genomic organization, 5 0 -upstream sequence, and chromosomal localization of an insulinoma-associated intronless gene, IA-1, J. Biol. Chem. 269 (1994) 14170–14174.
[7] K. Nichol, C.E. Pearson, CpG methylation modifies the genetic stability of cloned repeat sequences, Genome Res. 12 (2002) 1246– 1256. [8] T. Trinh, J. Jessee, F.R. Bloom, V. Hirtch, Stbl2, an E. coli strain for the stable propagation off retroviral clones and direct repeat sequences, Focus 16 (1994) 78–80. [9] H. Shizuya, B. Birren, U.J. Kim, V. Mancino, T. Slepak, Y. Tachiiri, M. Simon, Cloning and stable maintenance of 300kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector, Proc. Natl. Acad. Sci. USA 89 (1992) 8794–8797. [10] J. Wild, Z. Hradecna, W. Szybalski, Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones, Genome Res. 12 (2002) 1434–1444.
An effective alternate cloning strategy for unstable mouse genomic sequences Michael S. Lan *, Michelle Muguira The Research Institute for Children, ChildrenÕs Hospital, Departments of Pediatrics and Genetics, Louisiana State University Health Sciences Center, New Orleans, LA 70118, USA Received 25 February 2005 Available online 19 March 2005
Abstract Unstable mammalian genomic sequences frequently underwent spontaneous rearrangement during the bacterial cloning process. When the flanking sequences of an INSM1 gene comprised of 3.0 and 4.5 kb were subcloned into a targeting vector for a gene deletion study, both the genomic sequences underwent spontaneous rearrangement. Neither the usage of recombinase-free Escherichia coli competent cells nor lowering the culture incubation temperature averted the recombination events. Co-transformation of a methyltransferase vector, pAIT2, with the targeting vector had little effect in preventing recombination through methylation of the plasmid DNA. Here, we show that a single-copy cloning technique is effective to clone the unstable mouse genomic DNA into the targeting vector. Ó 2005 Elsevier Inc. All rights reserved. Keywords: INSM1; Recombination; Unstable genomic sequences; pBAC/oriV vector; Copy control cloning
The insulinoma-associated 1 (INSM1/IA-1) gene encodes a 510 amino acid zinc-finger transcriptional repressor originally isolated from a human insulinoma subtraction library [1,2]. Tissue-specific expression patterns revealed that the INSM1 gene is developmentally regulated in the pancreas and nervous system, as well as in tumors of neuroendocrine origin [3–5]. The INSM1 gene is located on human chromosome 20p11.2 and on mouse chromosome 2. Both were identified as intronless genes [4,6]. The flanking sequences of the INSM1 gene were first PCR amplified from the genomic DNA of the mouse strain 129/SVJ. The genomic organization of the INSM1 gene and restriction enzyme mapping were determined by sequence analysis. In order to elucidate the biological function of the INSM1 gene, a conditional targeting construct was designed for a gene knockout experiment. A pair of loxP *
Corresponding author. Fax: +1 504 896 2722. E-mail address: [email protected] (M.S. Lan).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.022
sites was designed to flank the partial INSM1 promoter sequence and the entire coding sequence. The 5 0 -arm (3.0 kb) and 3 0 -arm (4.5 kb) of the INSM1 genomic sequences were subsequently cloned into the flanking regions of the INSM1 gene for homologous recombination. In this study, both the 5 0 - and 3 0 -arm genomic sequences experienced spontaneous rearrangement during the bacterial cloning processes. After testing several cloning strategies, we succeeded in using a copy-control vector that limits the rearrangement events for these unstable genomic DNA sequences. This cloning technique could be of particular use when dealing with unstable genomic sequences during the construction of targeting vectors for gene knockout experiments.
Materials and methods FRT-loxP targeting vector construction. The FRT-loxP vector was kindly provided by Dr. C. Wright (Vanderbilt University, Nashville,
642
M.S. Lan, M. Muguira / Biochemical and Biophysical Research Communications 330 (2005) 641–644
TN). A HindIII fragment (3.5 kb) consisting of a partial promoter and the entire INSM1 coding sequence was subcloned into the FRT-loxP vector. The orientation of the insert was determined by sequence analysis. The 5 0 -arm (3.0 kb) and the 3 0 -arm (4.5 kb) of the INSM1 gene were PCR amplified with appropriate flanking enzyme cloning sites from 129/SVJ genomic DNA, using a high fidelity DNA polymerase (Invitrogen). Subsequent cloning processes were carried out using maximal efficient electrocompetent cells (XL10-Gold). Co-transformation of pAIT2 with the targeting vector for in vivo methylation. It has been reported that CpG methylation modifies the genetic stability of cloned repeat sequences [7]. In order to test the hypothesis that the CpG-rich region of the INSM1 gene may contribute to DNA instability, we employed a pAIT2 in vivo methylation system. Both the INSM1 targeting and pAIT2 vectors were co-transformed into Escherichia coli competent cells, and selected for both ampicillin and kanamycin resistant clones. Copy control single-copy cloning strategy. The very low copy bacterial artificial chromosome (BAC) vector is frequently used for its advantage of maintaining a high stability of inserted clones. A copy control PCR cloning kit (Epicentre) was used in our study. The FRTloxP-INSM1 fragment was digested with SalI/NotI, blunt-ended, and subcloned into the PCR cloning vector using a blue-white selection system. An electrocompetent TransforMax EPI300 E. coli that can be induced to a high-copy number within 5 h was used for this vector. The electroporation condition was modified to achieve high transformation efficiency since the vector size was over 15 kb. A conventional colony hybridization method was employed to select positive clones using the labeled insert as a probe.
Results and discussion The partial promoter and the entire coding sequence were HindIII cloned into the FRT-loxP targeting vector. However, the subsequent cloning of either the 5 0 -arm (3.0 kb) or 3 0 -arm (4.5 kb) sequences into either end of the targeting construct failed to generate a vector of the correct size (Fig. 1). The starting FRT-loxP vector, with the loxP sites flanking the promoter and the INSM1 coding sequence, represented a size of 11 kb. However, after the ligation of either the 5 0 -arm or 3 0 arm into the vector, the vector size was rearranged into 63.0 kb. Similar experimental results were obtained in many attempts of this simple cloning process. Therefore, we concluded that an unexpected recombination event must be occurring spontaneously during the cloning process. Analysis of the INSM1 flanking sequence and INSM1-3 0 untranslated sequence revealed tandem copies of CAn (n = 38) and CTn (n = 28) dinucleotide repeats, and the highly GC-rich sequences in the promoter region of the INSM1 gene. It is speculated that these repeat sequences could cause the recombination occurrence. In an effort to eliminate the recombination, two E. coli strains were selected, such as ElectroMAX Stbl4 (Invitrogen) and SURE2 (Stratagene), that reportedly improve the cloning capacity of repeat containing insert sequences [8]. The culture temperature was lowered to 25 °C, which should also help to prevent recombination. However, after screening over a hundred clones neither change showed any advantage
Fig. 1. Molecular cloning of the 5 0 -arm and 3 0 -arm sequences of the INSM1 gene caused rearrangement. The FRT-loxP-INSM1 vector (11 kb) was digested with SalI and ClaI, and ligated with the 5 0 -arm (3 kb, SalI/ClaI) overnight. The ligation mixture was transformed into XL10-Gold competent cells. Over a hundred colonies from at least five experiments were analyzed by SalI and ClaI digestion. A 2.8 kb band was present in all of the clones selected for analysis. Similarly, the FRT-loxP-INSM1 vector was digested with BamHI, treated with calf intestine phosphatase, and then ligated with the 3 0 -arm (4.5 kb, BamHI) overnight. The ligation mixture was transformed into XL10Gold competent cells. Over a hundred colonies from at lease five experiments were analyzed by BamHI digestion. Two bands, 3 and 2.2 kb, were present in the majority of the clones selected for analysis. The same results were observed in different experiments and are likely due to unstable genomic sequence rearrangement in the bacteria.
over the regular XL10-Gold competent cells that we normally use. We further evaluated whether CpG methylation could improve the genetic stability of the cloned repeat sequences [7]. The transformation was performed with the addition of an E. coli-based in vivo methylation system-pAIT2 (KanR). Two selection systems (AmpR and KanR) allowed the selective growth of cells containing both plasmids upon co-transformation. Since genomic stability is not solely dependent upon the presence of repeated sequences, co-transformation of the pAIT2 vector with the INSM1 genomic sequences did not inhibit the genomic sequence recombination (data not shown). An alternate strategy developed by Shizuya et al. [9] was based on the concept of using very low copy bacterial artificial chromosome vectors (BACs) for unstable genomic sequences. The disadvantage of a low copy number BAC vector was overcome by the development of a novel class of inducible single-copy/high-copy pBAC/oriV vectors [10]. This single-copy cloning technique could both greatly reduce the probability of recombination during the cloning process, and compensate for a low copy number by the conditional induction
M.S. Lan, M. Muguira / Biochemical and Biophysical Research Communications 330 (2005) 641–644
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Fig. 2. Construction of the INSM1 targeting vector into the copy-control pCC1 vector. The partial promoter and the entire INSM1 coding sequence (HindIII fragment, 3.5 kb) was cloned into a pFRT-loxP vector, named FRT-loxP-INSM1. The FRT-loxP-INSM1 fragment was digested with SalI and NotI, blunt-ended, and cloned into the copy-control pCC1 vector. The 5 0 -arm of the INSM1 gene (3 kb) was PCR amplified to include a ClaI site at both ends for pCC1 vector cloning. Subsequently, the 3 0 -arm of the INSM1 gene (4.5 kb) was PCR amplified with BamHI sites present at both ends for cloning into the FRT-loxP-INSM1 targeting vector. The electroporation voltage was modified by using a 0.1 cm cuvette with 1.5 kV setting. The orientation of each insert was determined by sequence analysis. The entire targeting sequence (15.5 kb) was digested with NotI sites from the copy-control pCC1 vector for ES cell transfection studies.
of the high-copy number through the trf A gene product supplied by the TransforMax EPI300 E. coli (Epicentre). This strategy was tested in our system by first blunt-end cloning the FRT-loxP-INSM1 (SalI/NotI fragment) into the pCC1 blunt-end cloning vector, using the blue-white selection method. The copy control clones can be induced to produce up to 200 plasmid copies per cell in 5 h [10]. This level of induction provides enough plasmid DNA for sequence analysis. The positive clones were subsequently subjected to the subcloning of the 5 0 -arm (ClaI site) followed by the 3 0 -arm (BamHI site) sequences into the copy-control pCC1 vector (Fig. 2). The cloning efficiency decreased drastically as the vector size increased to over 15 kb, and the electroporation condition had to be adjusted with a higher voltage setting. Positive clones were selected with a colony hybridization method. The cloning process was tedious due to the selection of unique restriction enzyme sites and the blunt-end ligation approach, but successfully eliminated the recombination event. Completion of the human genome project provides immense information for human genetics. The next task is to elucidate the biological function of each individual gene. Genetic instability always presents difficulties in laboratory manipulation of unstable genomic sequences. It is reported here that the flanking arm sequences of an intronless gene, INSM1, caused recombination, making them unclonable in bacteria. After testing various options, we showed that the commercially available copy-
control PCR cloning kit (Epicentre) provided an effective alternative for unstable genomic DNA cloning, which is essential for pursuing our gene targeting experiments.
Acknowledgments We thank Drs. S.H. Pincus, M.B. Breslin, M. Ferris, and D.S. Fox, for critical reading of the manuscript, Dr. B. Jack (New England BioLabs, Beverly, MA) for the pAIT2 vector, and Dr. C. Wright (Vanderbilt University, Nashville, TN) for the FRT-loxP vector. This work was supported by funds from the Research Institute for Children, ChildrenÕs Hospital, and a grant from NIDDK, National Institutes of Health (NIH) DK61436 (to M.S.L.).
References [1] Y. Goto, M.G. DeSilva, A. Toscani, B.S. Prabhakar, A.L. Notkins, M.S. Lan, A novel human insulinoma-associated cDNA, IA-1, encodes a protein with zinc-finger DNA-binding motifs, J. Biol. Chem. 267 (1992) 15252–15257. [2] M.B. Breslin, M. Zhu, A.L. Notkins, M.S. Lan, Neuroendocrine differentiation factor, IA-1, is a transcriptional repressor and contains a specific DNA-binding domain: identification of consensus IA-1 binding sequence, Nucleic Acids Res. 30 (2002) 1038– 1045.
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M.S. Lan, M. Muguira / Biochemical and Biophysical Research Communications 330 (2005) 641–644
[3] M.B. Breslin, M. Zhu, M.S. Lan, NeuroD1/E47 regulates the Ebox element of a novel zinc-finger transcription factor, IA-1, in developing nervous system, J. Biol. Chem. 278 (2003) 38991–38997. [4] J.P. Xie, T. Cai, H. Zhang, M.S. Lan, A.L. Notkins, The zincfinger transcription factor INSM1 is expressed during embryo development and interacts with the Cbl-associated protein, Genomics 80 (2002) 54–61. [5] M. Zhu, M.B. Breslin, M.S. Lan, Expression of a novel zincfinger cDNA, IA-1, is associated with rat AR42J cells differentiation into insulin-positive cells, Pancreas 24 (2002) 139–145. [6] M.S. Lan, Q. Li, J. Lu, W.S. Modi, A.L. Notkins, Genomic organization, 5 0 -upstream sequence, and chromosomal localization of an insulinoma-associated intronless gene, IA-1, J. Biol. Chem. 269 (1994) 14170–14174.
[7] K. Nichol, C.E. Pearson, CpG methylation modifies the genetic stability of cloned repeat sequences, Genome Res. 12 (2002) 1246– 1256. [8] T. Trinh, J. Jessee, F.R. Bloom, V. Hirtch, Stbl2, an E. coli strain for the stable propagation off retroviral clones and direct repeat sequences, Focus 16 (1994) 78–80. [9] H. Shizuya, B. Birren, U.J. Kim, V. Mancino, T. Slepak, Y. Tachiiri, M. Simon, Cloning and stable maintenance of 300kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector, Proc. Natl. Acad. Sci. USA 89 (1992) 8794–8797. [10] J. Wild, Z. Hradecna, W. Szybalski, Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones, Genome Res. 12 (2002) 1434–1444.