Elsevier

Gene

Volume 496, Issue 2, 1 April 2012, Pages 103-109
Gene

An arrayed human genomic library constructed in the PAC shuttle vector pJCPAC-Mam2 for genome-wide association studies and gene therapy

https://doi.org/10.1016/j.gene.2012.01.011Get rights and content

Abstract

The various iterations of the HapMap Project and many genome-wide association studies (GWAS) have identified hundreds of potential genes involved in monogenic and multifactorial traits. We constructed an arrayed 115,000-member human genomic library in the PAC shuttle vector pJCPAC-Mam2 that can be propagated in both bacterial and human cells. The library appears to represent a two-fold coverage of the human genome. Transient transfection of a p53-containing PAC clone into p53-null Saos-2 human osteosarcoma cells demonstrated that both p53 mRNA and protein were produced. Additionally, expression of the p53 protein triggered apoptosis in a subset of the Saos-2 cells. This library should serve as a valuable resource to validate potential disease genes identified by GWAS in human cell lines and in animal models. Also, individual library members could potentially be used for gene therapy trials for a variety of recessive disorders.

Highlights

► Human genomic PAC library for functional genomics. ► p53-containing PAC clone is transcribed in p53 null Saos-2 cells. ► p53-containing PAC clone is translated in p53 null Saos-2 cells. ► p53-containing PAC clone causes apoptosis in p53 null Saos-2 cells. ► Human genomic PAC library for gene therapy.

Introduction

The Human Genome Project has ushered in the era of big science. The International HapMap Project's original goal was to catalog the millions of single nucleotide polymorphisms (SNPs) present in many haplotypes in an attempt to establish links between certain variants and specific diseases. This project led to the identification of dense maps of human genetic variation. The International HapMap Project Phase 2 catalogued over 3 million polymorphisms (Sabeti et al., 2007). These investigations have uncovered over 150 risk loci for more than 60 common diseases and traits (Manolio and Collins, 2009). Genome-wide association studies (GWAS) are underway to identify candidate genes for many complex traits such as Alzheimer's disease, autism, bipolar disorder, Type I diabetes, multiple sclerosis, Parkinson's disease, obesity, rheumatoid arthritis and schizophrenia (Cichon et al., 2011, Menon and Farina, 2011, Stefansson et al., 2008a, Stefansson et al., 2008b, Thorliefsson et al., 2009, Weiss et al., 2008). As candidate genes are identified using a GWAS approach, investigators still need to demonstrate which if any genes have a causative role in the various diseases.

Genomic libraries constructed in the original BAC and PAC cloning systems were very useful for completion of the Human Genome Project (HGP). Libraries constructed in these vector systems were used to generate physical maps of all twenty three chromosome pairs and served as the templates for DNA sequencing (Ioannou et al., 1994, Kim et al., 1996, Shizuya et al., 1992). Construction of next generation BAC and PAC shuttle vectors that could be propagated in both bacterial and human cells allow scientists to study the functions of individual genes in tissue culture and in animal models. Inclusion of the Epstein Barr Virus latent origin of replication oriP allows for the episomal replication of these vectors in a broad range of mammalian cells including human, monkey, dog, murine, rodent and porcine cell lines (Black and Vos, 2002, Min et al., 2003, Miziguchi et al., 2000, Tomiyasu et al., 1998, Wolfgang et al., 2002, Yates et al., 1985). Libraries housed in the vectors pPAC4 and pBACe4 contained the EBV oriP required for propagation in mammalian cells, but both vectors lacked the EBNA1 gene that is necessary for episomal propagation of clones in mammalian cells. Therefore, investigators had to establish an EBNA1-expressing cell line before introducing a desired clone into human cells (Frengen et al., 2000). The shuttle vector pEBAC190G that was constructed later does contain the EBNA1 gene, but no human genomic libraries constructed in this vector have been reported (Al-Hasani et al., 2003). Additionally, none of these vector systems have the ability to activate a multi-copy origin of replication during propagation in Escherichia coli to increase the yield of individual library members to recover ample DNA for subsequent transfections into mammalian cells.

The shuttle vector pJCPAC-Mam1 contains two P1-derived origins of replication, the P1 single-copy replicon for low copy expression and a multi-copy lytic replicon under the control of the lac repressor for high copy expression upon the addition of IPTG in E. coli. This vector also contains the EBV latent replication origin oriP, the EBNA1 gene, and a puromycin-resistance gene for propagation of all library members in mammalian cells (Coren and Sternberg, 2001).

To increase the utility of this cloning vector a mutant loxP 5171 element was inserted into the unique NotI site of pJCPAC-Mam1 to generate pJCPAC-Mam2. Highly specific nested deletions could then be generated efficiently from both ends of any genomic DNA of interest using the mutant and wild-type loxP elements flanking the genomic insert (Chatterjee et al., 2004, Gundersen and Coren, 2007, Lee and Saito, 1998). This technology enables one to determine cis-acting sequences such as enhancers and repressors as well as to determine gene boundaries.

We constructed an arrayed human genomic library containing 115,000 clones in the PAC shuttle vector pJCPAC-Mam2. We digested a small subset of clones with the restriction enzyme NotI and then used FIGE to determine insert size. Additionally PCR screening of the final pools with nine genes was used to obtain an estimate of library coverage. A p53-containing PAC clone was transiently transfected into the p53-null Saos-2 human osteosarcoma cell line to demonstrate the utility of individual library members in human cell culture models. Members from this library can be used to validate candidate genes identified by GWAS in human cells and in animal models.

Section snippets

Insert size analysis and size distribution of random PAC clones

For the analysis of average genomic DNA insert size, 473 clones were randomly chosen from the 115,000-member library. Miniprep DNA was prepared from each clone, the DNA was digested with NotI and restriction fragment sizes were determined using FIGE. Since the vector contributes 23 kb, this value was subtracted from the size of each fragment(s) to obtain the insert size. The range of insert size was from 1 kb to 210 kb. Among all of the clones analyzed, 81.4% contained inserts, whereas 18.6%

Discussion

In this study an arrayed 115,000-member human genomic library was constructed in the PAC shuttle vector pJCPAC-Mam2. Investigators have previously constructed the RPCI-6 human genomic library in the PAC shuttle vector pPAC4 (Osoegawa et al, 2001). Unfortunately this vector lacks a multi-copy bacterial origin of replication that would significantly increase the yield of a given clone in E. coli. Also, pPAC4 lacks the EBNA1 gene needed to activate the EBV latent replication origin oriP.

Construction of the PAC library in pJCPAC-Mam2

A human genomic library was constructed in the PAC vector pJCPAC-Mam2 (version LoxP-1) (Gundersen and Coren, 2007). This vector was digested with BamHI and prepared as previously described (Coren and Sternberg, 2001). High molecular weight genomic DNA (Viromed passage six cells) was prepared from primary foreskin fibroblasts as previously described (Shepherd et al.,1994). Genomic DNA was partially digested with MboI and size fractionated through a 10-40% linear sucrose gradient. DNA in the

Acknowledgments

JSC would like to thank Jon Jarvik (SpectraGenetics) for providing a fosmid containing the human p53 gene; Robert Burke, Michael Wagner and Matthew Kochuba for their contributions in arraying the library, Heather Peluso for her contribution with the functional studies of p53 and Jessica Manchak and Laura Wingert for their technical assistance in subcloning the p53-GFP cassette into pJCPAC-Mam1. This work was supported by the National Institutes of Health [HG002216-01A1] and by Elizabethtown

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