Correct cell- and differentiation-specific expression of a murine alpha 1 (I) collagen minigene in vitro differentiating embryonal carcinoma cells

Abstract

An in vitro differentiation system utilizing retinoic acid (RA) treatment of pluripotent murine P19 embryonal carcinoma (EC) cells, which can be induced to differentiate into various cell types, was optimized for maximal induction of alpha 1 type I collagen (Col1a1) gene expression. Differentiation was associated with apoptotic death of the majority of cells, indicating that this in vitro system faithfully mimics the in vivo differentiation process. Col1a1 mRNA became detectable by RNase protection assay after 3 days of RA treatment and, after 6 days, reached a level comparable to that in NIH 3T3 fibroblasts. After induction of differentiation the Col1a1 gene remained transcriptionally active for extended periods of time even in the absence of RA. A minigene version of the murine Col1a1 gene was constructed that contains all of the so far known Col1a1 regulatory elements. This construct exhibited the correct expression pattern in stable transfection experiments: it was expressed in fibroblasts, but not in undifferentiated P19 EC cells, and it was transcriptionally activated after induction of differentiation. This experimental system should be a useful tool for dissecting the molecular mechanisms involved in the developmental activation and stage- and tissue-specific expression of the murine Col1a1 gene.

FIG. 3

RA-induced differentiation results in transcriptional activation of the murine Collal gene. Total RNA was isolated from 3T3 cells, undifferentiated P19 EC cells, and P19 EC cells treated with RA for the indicated lengths of time in days and analyzed by RNase protection assay using a probe that protects 112 nucleotides of Collal mRNA as described (9). (A) P19 EC cells were differentiated with RA for 1–6 days as described in the Materials and Methods section. On the original autoradiogram Collal mRNA was first detectable after 3 days of RA treatment. (B) P19 EC cells were differentiated with RA for 6 days and grown for 6 more days in the absence or presence of RA as indicated and analyzed by RNase protection assay. The concentration and integrity of the RNA preparations were determined by agarose gel electrophoresis (not shown), and equal amounts of RNA were assayed in each lane. With this probe we usually see in RNase protection assays a main band at 112 nucleotides and a lower band or smear, the intensity of which varies in different experiments (9,29,30). The origin of this lower band has not been determined, but it is assumed to result from inaccurate initiation of transcription and/or technical artefacts such as breathing of the hybrid molecules or unspecific degradation during RNase digestion.

FIG. 4

Schematic representation of the murine Collal minigene. The minigene used in these experiments contains 4 kb of 5′-flanking sequence, exons 1–6 from the 5′ end, exons 49–52 from the 3′ end, and 3 kb of 3′-flanking sequence. The 5′ and 3′ portions of the minigene were joined at intron sequences (a unique SnaB1 site in intron 6 and a blunted HindIII site in intron 48) to maintain correct splicing signals and translational reading frame. Unique restriction sites for Not 1 and Srf I were added at the 5′ and 3′ ends of the minigene, respectively, to allow subsequent addition of additional, putative remote regulatory elements as they are identified.

FIG. 5

RA-induced differentiation of P19 EC cells results in transcriptional activation of the endogenous Collal gene and the minigene. Total RNA was isolated from P19 EC and NIH 3T3 fibroblasts and analyzed by RNase protection assay using a probe that differentiates between the endogenous and minigene transcripts and a GAPDH probe as loading control (see the Materials and Methods section). The position of the GAPDH signal and the Collal endogenous and minigene signals are indicated by arrows. The integrity of the probes used in these assays is shown in the two rightmost lanes. (A) Minigene-transfected and untransfected NIH 3T3 fibroblasts. (B) Minigene-transfected, undifferentiated, and RA differentiated P19 cells analyzed 4 and 6 days after induction of differentiation as indicated; the cells analyzed in the 6d P#2 lane had been passed twice after induction of differentiation and show a relative higher expression of both the endogenous Collal gene and the minigene.