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

Cecilia Mancini,Erika Messana,Alessandro Brussino,Alfredo Brusco,Emilia Turco

doi:10.1186/1480-9222-13-10

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.

Highlights

To identify the mutant embryonic stem cells (ES) cell clones to be microinjected, two Southern blots are usually performed: one to detect ES clones in which homologous recombination has occurred, and the other to verify the number of neomycin phosphortransferase positive selection cassette (NEO) cassettes
It became necessary to have a rapid test to exclude the presence of additional copies of the NEO cassette in ES clones in which homologous recombination was successfully obtained
We describe a screening method using a rapid semi-quantitative real-time PCR, which was validated on ES clones with different NEO copies (0, 1, 2, > 2 copies), previously assessed by Southern blot

Summary

Introduction

* Correspondence: alfredo.brusco@unito.it 1Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy Full list of author information is available at the end of the article of the mutation present in the targeting construct into the gene of interest. To identify the mutant ES cell clones to be microinjected, two Southern blots are usually performed: one to detect ES clones in which homologous recombination has occurred, and the other to verify the number of NEO cassettes. It became necessary to have a rapid test to exclude the presence of additional copies of the NEO cassette in ES clones in which homologous recombination was successfully obtained.

Results

Conclusion