Rapid identification of homologous recombinants and determination of gene copy number with reference/query pyrosequencing (RQPS)

Abstract

Manipulating the mouse genome is a widespread technology with important applications in many biological fields ranging from cancer research to developmental biology. Likewise, correlations between copy number variations (CNVs) and human diseases are emerging. We have developed the reference-query pyrosequencing (RQPS) method, which is based on quantitative pyrosequencing and uniquely designed probes containing single nucleotide variations (SNVs), to offer a simple and affordable genotyping solution capable of identifying homologous recombinants independent of the homology arm size, determining the micro-amplification status of endogenous human loci, and quantifying virus/transgene copy number in experimental or commercial species. In addition, we also present a simple pyrosequencing-based protocol that could be used for the enrichment of homologous recombinant embryonic stem (ES) cells.

Second-generation Notch1 activity-trap mouse line (N1IP::CreHI) provides a more comprehensive map of cells experiencing Notch1 activity

“…Importantly, biochemical analysis indicates that this new allele is cleaved as efficiently as Notch1, supporting our previous observation that Notch trafficking relies on signals coded in the extracellular domain (Liu et al, 2013). This trap line, N1IP::Cre LO and the related sibling N1IP::Cre ERT2 (Liu et al, 2009;Pellegrinet et al, 2011) line, are powerful new tools for mapping alteration in Notch1 activation in vivo as a consequence of genetic or pharmaceutical intervention.…”

“…S2). This offers a simple, fast and non-radioactive method to pre-screen embryonic stem cells (ESCs) for homologous recombination with pyrosequencing (Liu et al, 2009), and enabled us to compare the abundance of mRNA produced by each allele (Liu et al, 2013 (V1744), thus normalizing the level of Cre to the NICD produced from the cleaved endogenous Notch1. Consistently, we found a twofold increase in the abundance of cleaved Cre relative to Cre6MT (Fig.…”

“…Cells were first pre-screened with pyroscreening, utilizing the C5178A polymorphism (Liu et al, 2009), and those with a C/A ratio between 40 and 60% at this position were further screened by both longrange PCR and Southern blot to confirm bona fide homologous recombinants [HR, (Liu et al, 2009); supplementary material Fig. S2)].…”

Notch1 loss of heterozygosity causes vascular tumors and lethal hemorrhage in mice

“…8), and the RosaLacZ (15) and RosaEYFP (16) reporter animals have been described previously. N1::Cre hi , an enhanced version of N1::Cre lo with an increased amount of N1::Cre fusion protein, is described previously (21,20) and in Supplemental Figure 1. RosaCBR, a Cre reporter line in which the gene encoding CBR luciferase is under the control of the ubiquitous Rosa26 promoter and preceded by a floxed stop cassette, is constructed in a similar way as the RosaLacZ (15) and RosaEYFP (16) knockin animals (Supplemental Figure 2).…”

Analysis of Transgene Integration

“…Accordingly, it becomes relevant to investigate the complexity of this array, the number of copies of the transgene integrated in the host genome. Several strategies have been developed in the field to estimate the number of copies, using initially Southern or dot-blot standard approaches (the latter relegated more to history) or, more commonly, using real-time quantitative PCR (qPCR) methods (Ballester et al, 2004;Mitrecić et al, 2005;Shepherd et al, 2009) or sometimes alternative pyrosequencing approaches (Liu et al, 2009).…”