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A library of mammalian effector modules for synthetic morphology.

Cachat E, Liu W, Hohenstein P, Davies JA - J Biol Eng (2014)

Bottom Line: Together with cell differentiation, these mechanisms allow populations of cells to organize themselves into defined geometries and structures, as simple embryos develop into complex organisms.Here we describe this library and demonstrate its use in the T-REx-293 human cell line to induce each of these desired morphological behaviours on command.Building on from the simple test systems described here, we want to extend engineered control of morphogenetic cell behaviour to more complex 3D structures that can inform embryologists and may, in the future, be used in surgery and regenerative medicine, making synthetic morphology a powerful tool for developmental biology and tissue engineering.

View Article: PubMed Central - PubMed

Affiliation: University of Edinburgh, Centre for Integrative Physiology, Hugh Robson Building, George Square, Edinburgh, EH8 9XD UK.

ABSTRACT

Background: In mammalian development, the formation of most tissues is achieved by a relatively small repertoire of basic morphogenetic events (e.g. cell adhesion, locomotion, apoptosis, etc.), permutated in various sequences to form different tissues. Together with cell differentiation, these mechanisms allow populations of cells to organize themselves into defined geometries and structures, as simple embryos develop into complex organisms. The control of tissue morphogenesis by populations of engineered cells is a potentially very powerful but neglected aspect of synthetic biology.

Results: We have assembled a modular library of synthetic morphogenetic driver genes to control (separately) mammalian cell adhesion, locomotion, fusion, proliferation and elective cell death. Here we describe this library and demonstrate its use in the T-REx-293 human cell line to induce each of these desired morphological behaviours on command.

Conclusions: Building on from the simple test systems described here, we want to extend engineered control of morphogenetic cell behaviour to more complex 3D structures that can inform embryologists and may, in the future, be used in surgery and regenerative medicine, making synthetic morphology a powerful tool for developmental biology and tissue engineering.

No MeSH data available.


Related in: MedlinePlus

The morphogenetic effectors used in this study.p27kip1 (control of proliferation), Casp2 (elective cell death), Cdh1 (cell-cell adhesion), p14 (fusion) and Crk-II (locomotion) were inserted downstream of a tetracycline-responsive promoter and coupled with different fluorescent reporters in pTREx destination vectors.
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Fig1: The morphogenetic effectors used in this study.p27kip1 (control of proliferation), Casp2 (elective cell death), Cdh1 (cell-cell adhesion), p14 (fusion) and Crk-II (locomotion) were inserted downstream of a tetracycline-responsive promoter and coupled with different fluorescent reporters in pTREx destination vectors.

Mentions: We assembled a Gateway® cloning (Invitrogen) compatible pENTR library of morphogenetic effectors: these include Cdh1 (cell-adhesion [13]), Casp2 (elective cell death [14], p14 (cell fusion [15]), Crk-II (cell locomotion [16]) and p27Kip1 (inhibition of otherwise constitutive proliferation [17]). The T-REx™-293 cell line (Invitrogen), a commercially available and easy-to-maintain cell line, was used to test these effector modules. It is derived from Human Embryonic Kidney (HEK) cells, to which stable expression of the tetracycline repressor protein has been added. T-REx-293 cells were used in combination with Gateway® pT-REx™-DEST vectors, so that driver genes could be conveniently inserted downstream of a tetracycline-inducible promoter from our library of pENTR library vectors. This way, effector modules could be switched on and off using tetracycline (Tet), without the need for construction and testing of the elaborate upstream logic modules that will control effector modules in advanced synthetic morphology projects [7, 8, 18]. After stable transfection, followed by clonal selection, independent clones were tested. Generally, effector genes were from a non-human origin in order to facilitate monitoring of their expression in T-REx-293 cells with species-specific antibodies or primers, the only exception being the gene encoding p27Kip1. Fluorescent reporters were also used in the system, by engineering pT-REx™-DEST vectors with IRES-TurboGFP or IRES-mCherry cassettes downstream of the effector, or by using 2A peptide fusions inside the effector cassette itself (Figure 1). Such reporters enable (i) the testing of clones for the simultaneous expression of the reporter and the gene of interest after stable selection and clonal isolation, (ii) real-time monitoring of inducible expression and (iii) visualization of different cell populations within a mixture of cells. Below, we demonstrate that each driver construct’s behaviour is effective in controlling the expected aspect of morphogenetic cell behaviour.Figure 1


A library of mammalian effector modules for synthetic morphology.

Cachat E, Liu W, Hohenstein P, Davies JA - J Biol Eng (2014)

The morphogenetic effectors used in this study.p27kip1 (control of proliferation), Casp2 (elective cell death), Cdh1 (cell-cell adhesion), p14 (fusion) and Crk-II (locomotion) were inserted downstream of a tetracycline-responsive promoter and coupled with different fluorescent reporters in pTREx destination vectors.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4255936&req=5

Fig1: The morphogenetic effectors used in this study.p27kip1 (control of proliferation), Casp2 (elective cell death), Cdh1 (cell-cell adhesion), p14 (fusion) and Crk-II (locomotion) were inserted downstream of a tetracycline-responsive promoter and coupled with different fluorescent reporters in pTREx destination vectors.
Mentions: We assembled a Gateway® cloning (Invitrogen) compatible pENTR library of morphogenetic effectors: these include Cdh1 (cell-adhesion [13]), Casp2 (elective cell death [14], p14 (cell fusion [15]), Crk-II (cell locomotion [16]) and p27Kip1 (inhibition of otherwise constitutive proliferation [17]). The T-REx™-293 cell line (Invitrogen), a commercially available and easy-to-maintain cell line, was used to test these effector modules. It is derived from Human Embryonic Kidney (HEK) cells, to which stable expression of the tetracycline repressor protein has been added. T-REx-293 cells were used in combination with Gateway® pT-REx™-DEST vectors, so that driver genes could be conveniently inserted downstream of a tetracycline-inducible promoter from our library of pENTR library vectors. This way, effector modules could be switched on and off using tetracycline (Tet), without the need for construction and testing of the elaborate upstream logic modules that will control effector modules in advanced synthetic morphology projects [7, 8, 18]. After stable transfection, followed by clonal selection, independent clones were tested. Generally, effector genes were from a non-human origin in order to facilitate monitoring of their expression in T-REx-293 cells with species-specific antibodies or primers, the only exception being the gene encoding p27Kip1. Fluorescent reporters were also used in the system, by engineering pT-REx™-DEST vectors with IRES-TurboGFP or IRES-mCherry cassettes downstream of the effector, or by using 2A peptide fusions inside the effector cassette itself (Figure 1). Such reporters enable (i) the testing of clones for the simultaneous expression of the reporter and the gene of interest after stable selection and clonal isolation, (ii) real-time monitoring of inducible expression and (iii) visualization of different cell populations within a mixture of cells. Below, we demonstrate that each driver construct’s behaviour is effective in controlling the expected aspect of morphogenetic cell behaviour.Figure 1

Bottom Line: Together with cell differentiation, these mechanisms allow populations of cells to organize themselves into defined geometries and structures, as simple embryos develop into complex organisms.Here we describe this library and demonstrate its use in the T-REx-293 human cell line to induce each of these desired morphological behaviours on command.Building on from the simple test systems described here, we want to extend engineered control of morphogenetic cell behaviour to more complex 3D structures that can inform embryologists and may, in the future, be used in surgery and regenerative medicine, making synthetic morphology a powerful tool for developmental biology and tissue engineering.

View Article: PubMed Central - PubMed

Affiliation: University of Edinburgh, Centre for Integrative Physiology, Hugh Robson Building, George Square, Edinburgh, EH8 9XD UK.

ABSTRACT

Background: In mammalian development, the formation of most tissues is achieved by a relatively small repertoire of basic morphogenetic events (e.g. cell adhesion, locomotion, apoptosis, etc.), permutated in various sequences to form different tissues. Together with cell differentiation, these mechanisms allow populations of cells to organize themselves into defined geometries and structures, as simple embryos develop into complex organisms. The control of tissue morphogenesis by populations of engineered cells is a potentially very powerful but neglected aspect of synthetic biology.

Results: We have assembled a modular library of synthetic morphogenetic driver genes to control (separately) mammalian cell adhesion, locomotion, fusion, proliferation and elective cell death. Here we describe this library and demonstrate its use in the T-REx-293 human cell line to induce each of these desired morphological behaviours on command.

Conclusions: Building on from the simple test systems described here, we want to extend engineered control of morphogenetic cell behaviour to more complex 3D structures that can inform embryologists and may, in the future, be used in surgery and regenerative medicine, making synthetic morphology a powerful tool for developmental biology and tissue engineering.

No MeSH data available.


Related in: MedlinePlus