Codon optimization markedly improves doxycycline regulated gene expression in the mouse heart

Area of Science:

• Molecular Biology
• Genetics
• Cardiovascular Research

Background:

• Tetracycline-regulated gene expression offers powerful control in transgenic animals.
• Constitutive transgenic expression in the heart causes perinatal lethality, necessitating regulated systems.
• Previous attempts using reverse tetracycline transactivator (rtTA) and transresponder (TR) systems faced challenges.

Purpose of the Study:

• To overcome perinatal lethality in transgenic hearts using tetracycline-inducible gene expression.
• To address limitations in rtTA expression driven by the cardiac alpha-myosin heavy chain promoter.
• To improve the regulation and efficiency of the tetracycline-inducible system in cardiac myocytes.

Main Methods:

• Utilized a tetracycline-regulated gene expression system with reverse tetracycline transactivator (rtTA) and transresponder (TR) lines.
• Investigated codon usage bias affecting rtTA expression under the cardiac alpha-myosin heavy chain promoter.
• Developed and tested a codon-optimized rtTA (opt-rtTA) construct.

Main Results:

• Identified codon usage bias as a major limitation for rtTA expression in cardiac myocytes.
• Co-injection of rtTA and TR transgenes resulted in unregulated TR gene expression in hemizygous animals.
• Codon optimization of rtTA significantly enhanced cardiac myocyte expression, increasing induction from 20-fold to 832-fold.
• Resulting opt-rtTA lines allow for homozygosity and efficient screening of doxycycline-regulated transgenes.

Conclusions:

• Codon optimization is crucial for effective rtTA expression in cardiac myocytes.
• The improved opt-rtTA system overcomes previous limitations, enabling robust doxycycline-regulated gene expression.
• This optimized system facilitates the generation of transgenic animals with tightly controlled cardiac gene expression for research.