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The cultivation of marine invertebrate cells in vitro has garnered significant attention due to the availability of diverse cell types and cellular potentialities in comparison to vertebrates and particularly in response to the demand for a multitude of applications. While cells in the colonial urochordate Botryllus schlosseri have a very high potential for omnipotent differentiation, no proliferating cell line has been established in Botryllus, with results indicating that cell divisions cease 24–72 h post initiation. This research assessed how various Botryllus blood cell types respond to in vitro conditions by utilizing five different refinements of cell culture media (TGM1–TGM5). During the initial week of culture, there was a noticeable medium-dependent increase in the proliferation and viability of distinct blood cell types. Within less than one month from initiation, we developed medium-specific primary cultures, a discovery that supports larger efforts to develop cell type-specific cultures. Specific cell types were easily distinguished and classified based on their natural fluorescence properties using confocal microscopy. These results are in agreement with recent advances in marine invertebrate cell cultures, demonstrating the significance of optimized nutrient media for cell culture development and for cell selection.
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Laboratory Culture and Mutagenesis of Amphioxus (Branchiostoma floridae).
Cephalochordates (amphioxus) are invertebrate chordates closely related to vertebrates. As they are evolving very slowly, they are proving to be very appropriate for developmental genetics studies aimed at understanding how vertebrates evolved from their invertebrate ancestors. To date, techniques for gene knockdown and overexpression have been developed, but methods for continuous breeding cultures and generating germline mutants have been developed only recently. Here we describe methods for continuous laboratory breeding cultures of the cephalochordate Branchiostoma floridae and the TALEN and Tol2 methods for mutagenesis. Included are strategies for analyzing the mutants and raising successive generations to obtain homozygotes. These methods should be applicable to any warm water species of cephalochordates with a relatively short generation time of 3-4 months and a life span of 3 years or more.
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- Award ID(s):
- 1952567
- PAR ID:
- 10395416
- Date Published:
- Journal Name:
- Methods in molecular biology
- Volume:
- 2219
- ISSN:
- 1940-6029
- Page Range / eLocation ID:
- 1-29
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1007/978-1-0716-0974-3_1
