Targeted gene disruption is a powerful tool for studying gene function in cells and animals. In addition, this technology includes a potential to correct disease-causing mutations. However, constructing targeting vectors is a laborious step in the gene-targeting strategy, even apart from the low efficiency of homologous recombination in mammals. Here, we introduce a quick and simplified method to construct targeting vectors. This method is based on the commercially available MultiSite Gateway® technology. The sole critical step is to design primers to PCR amplify genomic fragments for homologous DNA arms, after which neither ligation reaction nor extensive restriction mapping is necessary at all. The method therefore is readily applicable to embryonic stem (ES) cell studies as well as all organisms whose genome has been sequenced. Recently, we and others have shown that the human pre-B cell line Nalm-6 allows far high-efficiency gene targeting. The combination of the simplified vector construction system and the high-efficiency gene targeting in the Nalm-6 cell line has enabled rapid disruption of virtually any locus of the human genome within one month, and homozygous knockout clones lacking a human gene of interest can be created within 2–3 months. Thus, our system greatly facilitates reverse genetic studies of mammalian—particularly human—genes.

Introduction

Targeted gene disruption provides a powerful means for studying gene function by a reverse genetic approach (1,2). This technology depends on homologous recombination reactions that occur, albeit rarely, between transfected DNA (i.e., targeting vector) and the host genome. In mice, a large number of genes have been knocked out thus far using embryonic stem (ES) cells, and their physiological functions have been elucidated. More recently, reverse genetic studies using chicken DT40 cells have made a significant contribution to our understanding of cellular functions of a variety of genes (3). It is important to note, however, that the findings in model organisms are not always the case in human cells. Hence, reverse genetic analysis using human somatic cells would be of greater importance in the postgenome era when one attempts to reliably analyze the function of human genes. In human cultured cells, however, the frequency of gene targeting is typically too low for these approaches to be feasible (2,4). In this regard, it is noteworthy that we and others have shown that Nalm-6, a human pre-B acute lymphoblastic leukemia (ALL) cell line, is highly proficient in gene-targeting experiments (5,6,7,8).

A rate-limiting step in gene-targeting experiments, apart from the low efficiency of homologous recombination, is constructing targeting vectors. Vector construction typically involves cloning and mapping of genomic DNA fragments, which is highly time-consuming. Fortunately, the completion of the human genome sequence has greatly simplified those tedious processes, since virtually all DNA fragments can be readily PCR-amplified with specific primers (which must be designed not to include repetitive sequences such as Alu, however).

Nevertheless, the subsequent step—namely assembly of a genomic DNA fragment(s) with selectable markers—can still be a time-consuming and complicated process, partly due to the necessity of searching for appropriate restriction sites for ligation reactions as well as those for vector linearization. Thus, the success of vector construction depends crucially on the skill of the researcher. In this paper, we report a quick and simplified method for vector construction that we believe is simpler and quicker than any other methods reported thus far. We also show that virtually any genomic locus can be disrupted within one month in the human Nalm-6 cell line.

Materials and Methods

Vector Construction Using the MultiSite Gateway® System

To construct the pDEST DTA-MLS plasmid, a diphtheria toxin A (DT-A) gene cassette, which was excised from pMC1DT-ApA (Kurabo, Osaka, Japan) and a 58-mer multiple-linearization site (MLS) fragment carrying PacI, SwaI, I-SceI, AscI, and PmeI sites were sequentially inserted at the NdeI and AatII sites of pDEST^TM R4-R3, respectively (Invitrogen, Carlsbad, CA, USA; see Supplementary Figure S1 available online at www.BioTechniques.com). The 58-mer DNA fragment was made by annealing oligonucleotides MLS-A and MLS-B (Table 1).

Table 1. Oligonucleotides (Click to enlarge)

To create entry clones for floxed positive selectable markers hygromycin-resistance gene (Hyg^r), puromycin-resistance gene (Puro^r), and histidinol-resistance gene (His^r), each marker gene was subcloned into the donor vector pDONR™221, as illustrated in Supplementary Figure S2. Briefly, an entry clone plasmid, pENTR loxP, was first created by utilizing a DNA fragment generated by PCR with attB1- and attB2-containing primers (246-B1 and 246-B2, respectively; Table 1) using pBS246 (Gibco®; Invitrogen) as template. Floxed marker gene plasmids were constructed as depicted in Supplementary Figure S2, and each marker gene was subcloned into pENTR loxP at the NotI sites, yielding pENTR lox-Hyg, pENTR lox-Puro, and pENTR lox-His.

To generate targeting vectors for the human DNA ligase IV gene (LIG4), 2.2- and 2.0-kb LIG4 genomic fragments were obtained by PCR with ExTaq™ DNA polymerase (Takara Bio, Otsu, Japan) using Nalm-6 genomic DNA as template and were used as 5′ and 3′ arms, respectively. The primers used were L4-1 and L4-2 for the 5′ arm and L4-3 and L4-4 for the 3′ arm (see Table 1). By using the MultiSite Gateway system (more specifically, pENTR lox-Hyg, pENTR lox-Puro, and pDEST DTA-MLS), a floxed Hyg^r or Puro^r gene was inserted between the 5′ and 3′ arms on a plasmid carrying a DT-A gene, thus yielding targeting vectors pLIG4-Hyg and pLIG4-Puro. The targeting construct and knockout strategies for the KU80 gene have been described earlier (9).