Abstract
SUMMARYFluorescent proteins (FPs) have great utility in identifying specific cell populations and in studying cellular dynamics in the mouse. To quantify the factors that determine both the expression and relative brightness of FPs in mouse embryonic stem cells (mESCs) and in mice, we generated eight different FP-expressing ROSA26 alleles using recombinase-mediated cassette exchange (RMCE). These alleles enabled us to analyze the effects on FP expression of a translational enhancer and different 3′-intronic and/or polyadenylation sequences, as well as the relative brightness of five different FPs, without the confounding position and copy number effects that are typically associated with randomly inserted transgenes. We found that the expression of a given FP can vary threefold or more depending on the genetic features present in the allele. The optimal FP expression cassette contained both a translational enhancer sequence in the 5′-untranslated region (UTR) and an intron-containing rabbit β-globin sequence within the 3′-UTR. The relative expressed brightness of individual FPs varied up to tenfold. Of the five different monomeric FPs tested, Citrine (YFP) was the brightest, followed by Apple, eGFP, Cerulean (CFP) and Cherry. Generation of a line of Cherry-expressing mice showed that there was a 30-fold variation of Cherry expression among different tissues and that there was a punctate expression pattern within cells of all tissues examined. This study should help investigators make better-informed design choices when expressing FPs in mESCs and mice.
Highlights
Fluorescent proteins (FPs) are widely used to monitor gene expression and study cellular dynamics in cells and tissues (Tsien, 1989; Heim et al, 1994; Tsien and Miyawaki, 1998; Shaner et al, 2004)
In this study, we generated a ROSA26LCA allele and used it to create an allelic series of mouse embryonic stem cells (mESCs) to quantitatively assess both intrinsic and extrinsic variables that affect the relative brightness of FPs when expressed in mammalian cells and tissues
The pu tk fusion gene does not cause male infertility in transgenic animals (Chen and Bradley, 2000), thereby allowing LCAcontaining mESCs to be tested for germline competence. (2) pMCS.DT-A, a retrieval vector for gene targeting, contains an MC1-driven diphtheria toxin A sequence and multiple cloning site (MCS). (3) pMCS.66/2272 contains an MCS flanked by tandem lox66 and lox2272 sites that recombine with those in pLCA.71/2272 and can be used to generate exchange vectors for use in recombinase-mediated cassette exchange (RMCE). (4) pMCS.66/2272.Hygro is a variant of pMCS.66/2272 that contains a phosphoglycerol kinase (PGK)-driven hygromycin resistance gene flanked with tandem FLP recognition target (FRT) sites
Summary
Fluorescent proteins (FPs) are widely used to monitor gene expression and study cellular dynamics in cells and tissues (Tsien, 1989; Heim et al, 1994; Tsien and Miyawaki, 1998; Shaner et al, 2004). The successful use of FPs requires that their relative brightness in a cell exceeds the experimental detection threshold (Niswender et al, 1995) Many variables, both intrinsic and extrinsic to the FP, influence the brightness of FPs when expressed in cells or mice. Some of the intrinsic factors that influence FP brightness can readily be determined, extrinsic factors have been more difficult to quantify This is largely owing to the confounding effects of transgene copy number and the genetic insertion site, which can cause large variations in gene expression. The variability that occurs owing to transgene copy number and insertion site has been a major impediment in quantifying the genetic factors that influence FP reporter gene expression in cells and mice
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