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Main Authors:	Yoon, Jeongwoong, Tsuda, Taro, Bortoletto, Enrico, Sakaguchi, Akari, Kobayashi, Mutsuko, Rosani, Umberto, Yokoi, Hayato, Osada, Makoto, Venier, Paola, Nagasawa, Kazue
Format:	Artículo científico
Language:	en
Published:	Marine biotechnology (New York, N.Y.) 2025
Subjects:	Animals Hemocytes Transgenes Pectinidae Transfection Green Fluorescent Proteins Gene Expression
Online Access:	https://pubmed.ncbi.nlm.nih.gov/40498244/
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Simple Transgene Overexpression using Scallop Hemocyte Culture Platform Enables Functional Genetic Research in Molluscs. Yoon, Jeongwoong Tsuda, Taro Bortoletto, Enrico Sakaguchi, Akari Kobayashi, Mutsuko Rosani, Umberto Yokoi, Hayato Osada, Makoto Venier, Paola Nagasawa, Kazue Animals Hemocytes Transgenes Pectinidae Transfection Green Fluorescent Proteins Gene Expression Despite being evolutionarily and commercially important, molluscs have been a traditionally challenging group to study, due to their difficulty in maintenance under lab conditions and the lack of a genetic toolkit. Previously, we showed that transgene expression can be attained in molluscan cells with reporter genes under a molluscan virus promoter sequence. Following up, we developed a simple, efficient and rapid transgene expression platform using primary hemocyte culture of Farrer's scallop Chlamys farreri, a marine bivalve mollusc. The protocol consists of two steps: collection and seeding of hemocytes and incubation for 1 to 4 days with DNA-reagent mixture. We evaluated seven transfection reagents for three bivalve species and found that X-tremeGENE 360 was highly efficient for DNA transfection, particularly for C. farreri. Subsequently, C. farreri hemocyte culture and transfection conditions were examined, such as culture medium, size and form of DNA, and the mixing ratio of DNA and transfection reagent. Using this protocol, we visualized the subcellular localization of four bivalve oncogenes, Cf-Mdm2-like, Cf-c-Myc-like, Cf-Mortalin-like, and Cf-Ras-like, tagged with EGFP. Our hemocyte platform provides an easy entry to study cellular and molecular biology of molluscs and can be readily adapted for advanced methods such as live imaging and DNA-protein interaction assays, making the study of molluscs more accessible to the scientific community.

if (filemtime($filename) + $maxlifetime < time()) { unlink($filename); $count++; } } return $count; } /** * A function that is called internally when session data is to be saved. * * @param string $sessId The current session ID * @param string $data The session data to write * * @return bool */ protected function saveSession($sessId, $data): bool { $sessFile = $this->path . '/sess_' . $sessId; if ($handle = fopen($sessFile, 'w')) { $return = false; // Lock the file for exclusive access to avoid issues with multiple // processes writing session simultaneously: if (flock($handle, LOCK_EX)) { $return = fwrite($handle, $data); // Make sure that there's no trailing data by truncating the file to // the correct length: ftruncate($handle, strlen($data)); fflush($handle); flock($handle, LOCK_UN); } fclose($handle); if ($return !== false) { return true; } } // If we got this far, something went wrong with the file output... // It is tempting to throw an exception here, but this code is called // outside of the context of exception handling, so all we can do is // echo a message.

// Anecdotal testing Today and Yesterday seems to indicate destroy() // is called by the garbage collector and everything is good. // Something to keep in mind though. return 0; } /** * Write function that is called when session data is to be saved. * * @param string $sessId The current session ID * @param string $data The session data to write * * @return bool */ public function write($sessId, $data): bool { if ($this->writesDisabled) { return true; } return $this->saveSession($sessId, $data); } /** * A function that is called internally when session data is to be saved. * * @param string $sessId The current session ID * @param string $data The session data to write * * @return bool */ abstract protected function saveSession($sessId, $data): bool; }

* * @return void */ public function writeClose() { // The assumption is that we're using PHP's ext/session. // session_write_close() will actually overwrite $_SESSION with an // empty array on completion -- which leads to a mismatch between what // is in the storage object and $_SESSION. To get around this, we // temporarily reset $_SESSION to an array, and then re-link it to // the storage object. // // Additionally, while you _can_ write to $_SESSION following a // session_write_close() operation, no changes made to it will be // flushed to the session handler. As such, we now mark the storage // object isImmutable. $storage = $this->getStorage(); if (! $storage->isImmutable()) { $_SESSION = $storage->toArray(true); session_write_close(); $storage->fromArray($_SESSION); $storage->markImmutable(); } } /** * Attempt to set the session name * * If the session has already been started, or if the name provided fails * validation, an exception will be raised. * * @param string $name * @return SessionManager * @throws Exception\InvalidArgumentException */ public function setName($name) { if ($this->sessionExists()) { throw new Exception\InvalidArgumentException( 'Cannot set session name after a session has already started'

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