[Improvement of an in vitro transcription method in nuclei isolated from heart myocytes from control and hemodynamically overloaded rats].
The expression of genes crucial for different cardiac functions changes during ontogenic development and during hypertrophy due to hemodynamic overload. To determine if these changes are transcriptional or post-transcriptional, we describe here an optimized method to analyze in vitro the transcriptional activity of nuclei isolated from control and hemodynamically overloaded hearts of 23-day-old rats. Similar results were obtained with both types of hearts: (1) 3 to 6 million nuclei were isolated per gram of ventricular tissue; (2) nuclear incorporation of UTP32P increases with time, plateaus after about 10 to 20 min. and attains an average of 0.17 cpm per nucleus; (3) alpha-amanitin decreases total incorporation by 56%; (4) the sizes of the majority of transcribed RNA are between 0.2 and 4 kb, although a significant fraction of high molecular weight transcripts are also seen (between 4 and 6 kb). All this indicates that mRNAs are transcribed to a significant extent, suggesting that it is now possible, given appropriate nucleotide probes, to analyze the transcription of any given gene during cardiac growth.
Chassagne
C
C
I.N.S.E.R.M. Unité n. 127, Hôpital Lariboisière, Paris.
Boheler
K R
KR
Schwartz
K
K
D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry*; D005260:Female; D006439:Hemodynamics / Q000235:genetics; D066298:In Vitro Techniques; D009206:Myocardium / Q000166:cytology*; D015138:RNA, Nuclear / Q000235:genetics*; D051381:Rats; D011919:Rats, Inbred Strains; D014158:Transcription, Genetic
D015138:RNA, Nuclear
Mise au point d'une méthode de transcription in vitro dans des noyaux isolés de myocytes de coeurs de rats contrôles et hémodynamiquement surchargés.