Role of domains 3-4 (d3-4) of connective tissue growth factor (CTGF/CCN2) in lung fibrosis
In this project we examine the function of a carboxyl-terminal fragment of "connective tissue growth factor" (CTGF/CCN2) in vivo. Connective tissue growth factor (CTGF/CCN2) is implicated in several diseases, and is thought to be a key mediator of tissue fibrosis. We previously found that overexpression of full length CTGF/CCN2 have minimal implications on cardiac fibrosis in mice. However,we recently disclosed that full-length CTGF/CCN2 is inactive and must undergo proteolytic cleavage in order to release its biologically active entity, the carboxyl-terminal fragment comprised of domains III-IV (d3-4-CTGF).
We have established genetically engineered mouse with a floxed stop signal (loxP-STOP-loxP) for initiation of transcription of d3-4-CTGF(LSLd34CTGF-IRES-GFP) (FOTS:19482). The transgene (LSLd34CTGF) has been inserted into the ROSA26 locus where it remains inactive until the STOP signal in the promoter is cleaved out by Cre recombinase. Therefore, the mouse model can be used for the targeted conditional expression of d3-4-CTGF in any tissue that produce Cre-recombinase enzyme.
Here, we aim to establish expression of d3-4-CTGF transgene in the lungs by delivering replication deficient Adeno virus encoding Cre-recombinase(Adeno-Cre) into the lungs through intranasal inhalation, oropharyngeal aspiration or intratracheal intubation. Exposure of the loxP sites to Cre recombinase in the lung tissue would facilitate DNA recombination leading to overexpression of CTGF domains-III-IV of in a tissue specific manner. This will let us investigate the effect of transgenic overexpression of bioactive d3-4-CTGF in lung tissue in these mice and whether d3-4-CTGF is sufficient to produce pro-fibrotic properties reported for CTGF. This would further implicate on how CTGF may be targeted therapeutically.
Distress: For induction of transgene, we will administer a single dose of Adeno-Cre into anesthetized mice either by intranasal-inhalation, oropharyngeal or intratracheal method. We will euthanize mice at different time points post-administration (2wk/4wk/6wk) and harvest lungs for biochemical and histological analysis. The procedure will inflict minor transient distress on the animals. Based on previous studies which show over expression of full-length CTGF in the lungs may lead to fibrogenic responses or fibrosis, we can anticipate similar effects from the over expression of bioactive d-3-4-CTGF in this study. Therefore we will carefully monitor the mice after Adeno-Cre instillation to uncover the defined humane endpoint that may potentially occur before the scheduled termination of the experiment.
Number of animals:216
3R:We will use mice generated from the breeding plan of our LSLd34CTGF colony (described in FOTS 19482). The use of an adenovirus to deliver the Cre-recombinase circumvents the need for complex breeding with Cre-recombinase expressing strains (necessary for expression in most other organs) and will thus represent a significant reduction in the number of animals required to answer our questions. We will test the efficiency of our method of administration in a pilot study with a control virus before start of the actual study. All animal experiments will be conducted by well-trained project participants. Any distress due to transgene expression in these mice will be monitored and used to determine humane endpoints. Together these will facilitate adhering to 3R.
We have established genetically engineered mouse with a floxed stop signal (loxP-STOP-loxP) for initiation of transcription of d3-4-CTGF(LSLd34CTGF-IRES-GFP) (FOTS:19482). The transgene (LSLd34CTGF) has been inserted into the ROSA26 locus where it remains inactive until the STOP signal in the promoter is cleaved out by Cre recombinase. Therefore, the mouse model can be used for the targeted conditional expression of d3-4-CTGF in any tissue that produce Cre-recombinase enzyme.
Here, we aim to establish expression of d3-4-CTGF transgene in the lungs by delivering replication deficient Adeno virus encoding Cre-recombinase(Adeno-Cre) into the lungs through intranasal inhalation, oropharyngeal aspiration or intratracheal intubation. Exposure of the loxP sites to Cre recombinase in the lung tissue would facilitate DNA recombination leading to overexpression of CTGF domains-III-IV of in a tissue specific manner. This will let us investigate the effect of transgenic overexpression of bioactive d3-4-CTGF in lung tissue in these mice and whether d3-4-CTGF is sufficient to produce pro-fibrotic properties reported for CTGF. This would further implicate on how CTGF may be targeted therapeutically.
Distress: For induction of transgene, we will administer a single dose of Adeno-Cre into anesthetized mice either by intranasal-inhalation, oropharyngeal or intratracheal method. We will euthanize mice at different time points post-administration (2wk/4wk/6wk) and harvest lungs for biochemical and histological analysis. The procedure will inflict minor transient distress on the animals. Based on previous studies which show over expression of full-length CTGF in the lungs may lead to fibrogenic responses or fibrosis, we can anticipate similar effects from the over expression of bioactive d-3-4-CTGF in this study. Therefore we will carefully monitor the mice after Adeno-Cre instillation to uncover the defined humane endpoint that may potentially occur before the scheduled termination of the experiment.
Number of animals:216
3R:We will use mice generated from the breeding plan of our LSLd34CTGF colony (described in FOTS 19482). The use of an adenovirus to deliver the Cre-recombinase circumvents the need for complex breeding with Cre-recombinase expressing strains (necessary for expression in most other organs) and will thus represent a significant reduction in the number of animals required to answer our questions. We will test the efficiency of our method of administration in a pilot study with a control virus before start of the actual study. All animal experiments will be conducted by well-trained project participants. Any distress due to transgene expression in these mice will be monitored and used to determine humane endpoints. Together these will facilitate adhering to 3R.