Gene-targeted embryonic stem cells: real-time PCR assay for estimation of the number of neomycin selection cassettes

Abstract

In the preparation of transgenic murine ES cells it is important to verify the construct has a single insertion, because an ectopic neomycin phosphortransferase positive selection cassette (NEO) may cause a position effect. During a recent work, where a knockin SCA28 mouse was prepared, we developed two assays based on Real-Time PCR using both SYBR Green and specific minor groove binder (MGB) probes to evaluate the copies of NEO using the comparative delta-delta Ct method versus the Rpp30 reference gene.We compared the results from Southern blot, routinely used to quantify NEO copies, with the two Real-Time PCR assays. Twenty-two clones containing the single NEO copy showed values of 0.98 ± 0.24 (mean ± 2 S.D.), and were clearly distinguishable from clones with two or more NEO copies.This method was found to be useful, easy, sensitive and fast and could substitute for the widely used, but laborious Southern blot method.

Figure 1

Real-time PCR protocols for MGB- (left) and SYBR-assay (right). Protocols and run conditions that were used for MGB- and SYBR- assays are described in the upper part. Below are the oligo sequences and oligo concentrations used to prepare the NEO and Rpp30 assays. For the MGB-assay we prepared two (20×) mixes, one (*) for the target gene (NEO) and one (**) for the reference gene (Rpp30). For both assays, the final concentration in each reaction was 900 nM for oligos, 250 nM for probes (MGB-assay only). The MGB assay was prepared using TaqMan 2× Master Mix (Applied Biosystems): a standard run protocol of two hours was used. The SYBR-assay was performed with Fast SYBR Green 2× Master Mix (Applied Biosystems); the fast protocol reported took only 40 min.

Figure 2

Quantitative real-time PCR validation and results for NEO copy number determination, using MGB-assay (in red) and SYBR-assay (in green). (A) Twenty-two ES clones (abscissa, numbers from 1 to 22) showed a nNEO value between 0.8 and 1.2 (ordinate). One genomic DNA from a homozygous knockout mouse was used as control (CTRL 2NEO, nNEO = 2.2). Error bars indicate triplicates delta-delta Ct standard deviations. (B) Real-time PCR results: 45 ES clones are plotted on the graph, for each assay using a log₂ scale. The dotted line indicates a nNEO threshold of 1.2, the upper cutoff for considering the presence of a single NEO copy. Samples tested with either of the two assays had similar nNEO values, as shown by the similar population plots.