Name: generation of genetically modified mice through the microinjection of oocytes
Uploaded: 2017-06-15T13:00:00.000+00:00
Duration: 10 min 19 s
Description: Watch this Scientific Journal Video about Generation of Genetically Modified Mice through the Microinjection of Oocytes at JoVE.com

המחברים מודים לצוות של מתקן החיות (BRC) על תמיכתם המתמשכת. עבודה זו מומנה על ידי המועצה הלאומית למחקר רפואי רפואי ומועצת המחקר האוסטרלית.

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המחברים מספקים שירותי transgenesis אקדמי בעכברים באמצעות אוניברסיטת ניו סאות &#39;ויילס מארק ויינרייט אנליטית מרכז.

צעדים קריטיים בתוך הפרוטוקול הדור של עכברים מהונדסים גנטית ידוע להיות מאתגר מבחינה טכנית. עם זאת, הפרוטוקול המוצג כאן היא שיטה אופטימיזציה ופשוט המאפשר אחד לשלוט ולפתור את הטכניקה בזמן שיא. ישנם שני שלבים הכרחיים להשלמת הטכניקה ב?...

מודלים של בעלי חיים, הן בחולייתנים והן בחסרי חוליות, שימשו כלי עזר לבחינת הפתופיזיולוגיה של מצבים אנושיים כגון מחלת אלצהיימר 1 , 2 . הם גם כלים שלא יסולאו בפז כדי לחפש מחליפי מחלות ובסופו של דבר לפתח אסטרטגיות טיפול חדש בתקווה של תרופה. למרות שלכל מודל יש מגבלות פנימיות, השימוש בבעלי חיים כמודלים מערכתיים שלמים הוא חיוני למחקר ביו-רפואי. הסיבה לכך היא כי הסביבה הפיזיולוגית מטבולית ומורכבת לא ניתן לדמות לחלוטין בתרבית רקמות. עד כה, העכבר נשאר המין היונקים הנפוצים ביותר המשמשים מניפולציה גנטית כי זה כולל מספר יתרונות. תהליכים פיזיולוגיים וגנים הקשורים למחלות הם שמורים מאוד בין עכברים ובני אדם. העכבר היה יונק הראשון יש רצף הגנום המלא שלה (2002), שנה אחת לפני geno האדםלי (2003). מלבד זה עושר של מידע גנטי, העכבר יש יכולות הרבייה טובה, מחזור פיתוח מהיר (6 שבועות מן ההפריה לגמילה), וגודל סביר. כל היתרונות הללו, בשילוב עם אינדיקטורים פיזיולוגיים, כגון צבעי מעיל נפרדים (נדרש לחצות אסטרטגיות), הפכו את העכבר למודל אטרקטיבי עבור מניפולציה גנטית. יש לציין, בגיל מוקדם מאוד של גנטיקה מודרנית, גרגור מנדל התחיל לעבוד על עכברים לפני המעבר צמחים 3 . טכניקות העברת גנים גרמו לדור של העכבר המהונדס הראשון מעל לפני שלושה עשורים 4 , נוצר בתחילה באמצעות משלוח ויראלי. עם זאת, החוקרים הבינו עד מהרה כי אחד האתגרים העיקריים של transgenesis העכבר היה חוסר היכולת לשלוט על גורלו של ה- DNA אקסוגני. בגלל המסירה ויראלי של transgenes לתוך הביציות העכבר הביא עותקים מרובים משולבים באופן אקראי לתוך הגנום, possibilitY של הקמת קווים מהונדסים שלאחר מכן היה מוגבל. מגבלה אחת כזו היתה להתגבר כאשר גורדון et al. שנוצר קו העכבר מהונדס הראשון על ידי microinjection 5 , 6 . זה התחיל את העידן של טכנולוגיית DNA רקומביננטי, ואת הפרמטרים המשפיעים על התוצאה של הפגישה microinjection נחקרו באופן נרחב 7 . למרות microinjection אינו מאפשר שליטה על אתר האינטגרציה של transgene (אשר בסופו של דבר תוצאות רמות ביטוי ספציפי עבור כל עכבר מייסד), היתרון העיקרי של microinjection pronuclear נשאר היווצרות של concatemers ( כלומר, מערכים של עותקים מרובים של transgene, מקושר בסדרה) לפני שילוב גנומי 5 . מאפיין זה שימש במשך השנים להקים אלפי שורות עכבר מהונדס כי overexpress גן עניין. מאז, transgenesis, אשינוי מלאכותי של הגנום של האורגניזם, נעשה שימוש נרחב כדי לזהות את התפקיד של גנים בודדים בהתרחשות של מחלות. הישג מפתח נוסף על מניפולציה של הגנום העכבר הגיע כאשר מריו Capecchi בהצלחה שיבשו גן אחד בעכבר, פתיחת עידן של הגן מיקוד 8 . עם זאת, החסרונות העיקריים שהגיחו במהירות ממיקוד גנים המבוסס על תאי גזע ES, כולל האתגרים של תאי ES מתורבתים, מידת השתנות מסוימת של קימברליות ואורך התהליך ( כלומר, 12-18 חודשים, מינימום, כדי להשיג את העכבר) . לאחרונה, ההתקדמות בטכנולוגיות חדשות, כגון אנדונואלאסים מהונדסים ( למשל, נוקלאזות אצבע אבץ (ZFN), גרעיני אפקט מניע כמו activator (TALEN), וקבוצות חוזרות ונשנות של פליינדרומיס מקובצים בקביעות (CRISPR / Cas9) התפתחו כשיטות חלופיות להאיץ את תהליך המיקוד הגנטי ב- micE 9 , 10 . אלה endonucleases יכול בקלות להיות מוזרק לתוך ביציות העכבר על ידי microinjection, המאפשר לדור של עכברים ממוקד תוך פחות מ 6 שבועות. מאז הדו&quot;ח הראשון על השימוש CRISPR עבור עריכת הגנום 11 , מערכת חיסונית חיידקית אדפטיבית זו החליפה ZFN ו TALEN בשל היתרונות הרבים שלה, כולל את הקלות של סינתזה ואת היכולת למקד לוקוסים מרובים בבת אחת (המכונה &quot;ריבוב&quot; &quot;). CRISPR שימש לראשונה עבור מיקוד גנים בעכברים 12 ומאז הוחל על מינים אינספור, מן הצמחים לבני אדם 13 , 14 . עד כה, אין דיווח על מין אחד עמיד לעריכת הגנום CRISPR. שני השלבים המגבילים העיקריים של הדור של עכברים מהונדסים הם הזרקה של ביציות ואת ההשתלה מחדששל הביציות האלה לתוך נקבות מדומות בהיריון. למרות טכניקה זו תוארה על ידינו 15 ועוד 16 , שיפורים טכניים האחרונות עכבר embryology וטכניקות העברת הגן יש מהפכה בתהליך של יצירת עכברים מהונדסים גנטית. שיפורים אלה יתוארו כאן.

<table><tbody><tr><td>Micropipette 0.1-2.5 &amp;mu;L</td><td>Eppendorf</td><td>4920000016</td><td></td></tr><tr><td>Micropipette 2 - 20 &amp;mu;L</td><td>Eppendorf</td><td>4920000040</td><td></td></tr><tr><td>Micropipette 20 - 200 &amp;mu;L</td><td>Eppendorf</td><td>4920000067</td><td></td></tr><tr><td>Micropipette 100 - 1,000 &amp;mu;L</td><td>Eppendorf</td><td>4920000083</td><td></td></tr><tr><td>Molecular weight marker&amp;nbsp;</td><td>Bioline</td><td>BIO-33025</td><td>HyperLadder 1 kb</td></tr><tr><td>Molecular weight marker&amp;nbsp;</td><td>Bioline</td><td>BIO-33056</td><td>HyperLadder 100 bp</td></tr><tr><td>Agarose</td><td>Bioline</td><td>BIO-41025</td><td></td></tr><tr><td>EDTA buffer</td><td>Sigma-Aldrich</td><td>93296</td><td>10x - Dilute to 1x</td></tr><tr><td>Ethidium bromide</td><td>Thermo Fisher Scientific</td><td>15585011</td><td></td></tr><tr><td>SYBR Safe gel stain</td><td>Invitrogen</td><td>S33102</td><td></td></tr><tr><td>Gel extraction kit</td><td>Qiagen</td><td>28706</td><td></td></tr><tr><td>PCR purification kit (Qiaquick)</td><td>Qiagen</td><td>28106</td><td></td></tr><tr><td>Vacuum system (Manifold)</td><td>Promega</td><td>A7231</td><td></td></tr><tr><td>Nuclease-free microinjection buffer&amp;nbsp;</td><td>Millipore</td><td>MR-095-10F</td><td></td></tr><tr><td>Ultrafree-MC microcentrifuge filter&amp;nbsp;</td><td>Millipore</td><td>UFC30GV00</td><td></td></tr><tr><td>Cas9 mRNA</td><td>Sigma-Aldrich</td><td>CAS9MRNA</td><td></td></tr><tr><td>CRISPR expressing plasmid (px330)</td><td>Addgene</td><td>42230</td><td></td></tr><tr><td>Nuclease free water</td><td>Sigma-Aldrich</td><td>W4502</td><td></td></tr><tr><td>Phusion polymerase</td><td>New England Biolabs</td><td>M0530L</td><td></td></tr><tr><td>T7 Quick High Yield RNA kit</td><td>New England Biolabs</td><td>E2050S</td><td></td></tr><tr><td>RNA purification spin columns (NucAway)</td><td>Thermo Fisher Scientific</td><td>AM10070</td><td></td></tr><tr><td>ssOligos</td><td>Sigma-Aldrich</td><td>OLIGO STANDARD</td><td></td></tr><tr><td>Donor plasmid</td><td>Thermo Fisher Scientific</td><td>GeneArt</td><td></td></tr><tr><td>Hyaluronidase</td><td>Sigma-Aldrich</td><td>H3884</td><td></td></tr><tr><td>KSOMaa embryo culture medium</td><td>Zenith Biotech&amp;nbsp;</td><td>ZEKS-100</td><td></td></tr><tr><td>Mineral oil</td><td>Zenith Biotech&amp;nbsp;</td><td>ZSCO-100</td><td></td></tr><tr><td>M2 Medium</td><td>Sigma-Aldrich</td><td>M7167</td><td></td></tr><tr><td>Cytochalasin B</td><td>Sigma-Aldrich</td><td>C6762</td><td></td></tr><tr><td>Mouthpiece</td><td>Sigma-Aldrich</td><td>A5177</td><td></td></tr><tr><td>Glass microcapillaries</td><td>Sutter Instrument</td><td>BF100-78-10</td><td></td></tr><tr><td>Proteinase K</td><td>Applichem</td><td>A3830.0100</td><td></td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools&amp;nbsp;</td><td>91150-20</td><td></td></tr><tr><td>Iris scissors</td><td>Fine Science Tools&amp;nbsp;</td><td>91460-11</td><td></td></tr><tr><td>Vessel clamp</td><td>Fine Science Tools&amp;nbsp;</td><td>18374-43</td><td></td></tr><tr><td>Wound clips&amp;nbsp;</td><td>Fine Science Tools&amp;nbsp;</td><td>12040-01</td><td></td></tr><tr><td>Clips applier&amp;nbsp;</td><td>Fine Science Tools&amp;nbsp;</td><td>12018-12</td><td></td></tr><tr><td>Micro-scissors&amp;nbsp;</td><td>Fine Science Tools&amp;nbsp;</td><td>15000-03</td><td></td></tr><tr><td>Cauterizer</td><td>Fine Science Tools&amp;nbsp;</td><td>18000-00</td><td></td></tr><tr><td>Non-absorbable surgical sutures (Ethilon 3-0)</td><td>Ethicon</td><td>1691H</td><td></td></tr><tr><td>5% CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>MG Scientific</td><td>Galaxy 14S</td><td></td></tr><tr><td>Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>Nanodrop 2000c</td><td></td></tr><tr><td>Thermocycler</td><td>Eppendorf</td><td>6321 000.515</td><td></td></tr><tr><td>Electrophoresis set up</td><td>BioRad</td><td>1640300</td><td></td></tr><tr><td>UV Transilluminator</td><td>BioRad</td><td>1708110EDU</td><td></td></tr><tr><td>Thermocycler</td><td>Eppendorf</td><td>6334000069</td><td></td></tr><tr><td>Stereoscopic microscope</td><td>Olympus</td><td>SZX7</td><td></td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>IX71</td><td></td></tr><tr><td>2x Micromanipulators</td><td>Eppendorf</td><td>5188000.012</td><td></td></tr><tr><td>Oocytes manipulator</td><td>Eppendorf</td><td>5176000.025</td><td></td></tr><tr><td>Microinjector (Femtojet)</td><td>Eppendorf</td><td>5247000.013</td><td></td></tr><tr><td>Mice C57BL/6J strain</td><td>Australian BioResources</td><td>C57BL/6JAusb&amp;nbsp;</td><td></td></tr></tbody></table>

generation of genetically modified mice through the microinjection of oocytes

השימוש בעכברים מהונדסים גנטית תרם באופן משמעותי למחקרים על תהליכים פיזיולוגיים ופתולוגיים בתהליכי vivo . הזרקת pronuclear של ביטוי דנ&quot;א בונה לתוך ביציות מופרות נשאר הטכניקה הנפוצה ביותר לייצר עכברים מהונדס overexpression. עם כניסתה של טכנולוגיית CRISPR עבור מיקוד גנים, הזרקת pronuclear לתוך ביציות מופרות הורחב לדור של שני נוקאאוט ועכברים נוקין. עבודה זו מתארת ​​את הכנת ה- DNA להזרקה ואת הדור של מדריכים CRISPR עבור מיקוד גנים, עם דגש מיוחד על בקרת איכות. ההליכים הגנוטיפיים הדרושים לזיהוי המייסדים הפוטנציאליים הם קריטיים. אסטרטגיות גנוטיפינג חדשניות המנצלות את יכולות ה&quot;רב-ריבוב &quot;של CRISPR מוצגות כאן. הליכים כירורגיים מתוארים גם. יחד, השלבים של הפרוטוקול יאפשר את הדור של genעכברים מותאמים באופן מהותי עבור הקמתם הבאים של מושבות עכבר עבור שפע של תחומי מחקר, כולל אימונולוגיה, מדעי המוח, סרטן, פיזיולוגיה, פיתוח, ועוד.

להלן, זרימות עבודה עבור microinjection במקרה של אינטגרציה אקראית CRISPR בתיווך גנים מיקוד מתוארים ( איור 1 ). <img alt="איור 1" class="xfigimg" src="/files/ftp_upload/55765/55765fig1v2.jpg" /> איור 1: זרימת עבודה אופי?...

Watch this Scientific Journal Video about Generation of Genetically Modified Mice through the Microinjection of Oocytes at JoVE.com

Generation of Genetically Modified Mice through the Microinjection of Oocytes

Microinjection של ביציות העכבר משמש בדרך כלל עבור transgenesis קלאסי ( כלומר, שילוב אקראי של transgenes) ו CRISPR בתיווך גישור המיקוד. פרוטוקול זה סוקר את ההתפתחויות האחרונות microinjection, עם דגש מיוחד על בקרת איכות אסטרטגיות genotyping.

הדור של עכברים השתנה גנטית דרך Microinjection של ביציות

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

generation-genetically-modified-mice-through-microinjection

Research

JoVE Journal

Developmental Biology

20.5K Views.  University of New South Wales.  Microinjection של ביציות העכבר משמש בדרך כלל עבור transgenesis קלאסי ( כלומר, שילוב אקראי של transgenes) ו CRISPR בתיווך גישור המיקוד. פרוטוקול זה סוקר את ההתפתחויות האחרונות microinjection, עם דגש מיוחד על בקרת איכות אסטרטגיות genotyping. 

Video: Generation of Genetically Modified Mice through the Microinjection of Oocytes

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

Loss- ו-של-פונקצית רווח גישה לחקר תא מוקדם קובעי גורל preimplantation עכבר עובר

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

באמצעות העכבר Oocytes כדי להעריך תפקוד הגן האנושי במהלך המיוזה אני

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Genetics

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

Lentiviral מתווכת הייצור של העכברים הטרנסגניים: שיטה פשוטה ויעילה מאוד עבור לימוד ישיר של המייסדים

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

השימוש של עוברים הקפאת-הפשפה לייצור יעילות גבוהה של עכברים ששונו גנטית

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...

Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation

Germline genetically engineered mouse models (G-GEMMs) have provided valuable insight into in vivo gene function in development, homeostasis, and disease. However, the time and cost associated with colony creation and maintenance are high. Recent advances in CRISPR-mediated genome editing have allowed the generation of somatic GEMMs (S-GEMMs) by directly targeting the cell/tissue/organ of interest.
The oviduct, or fallopian tube in humans, is considered the tissue-of-origin of the most common ovarian cancer, high-grade serous ovarian carcinomas (HGSCs). HGSCs initiate in the region of the fallopian tube distal to the uterus, located adjacent to the ovary, but not the proximal fallopian tube. However, traditional mouse models of HGSC target the entire oviduct, and thus do not recapitulate the human condition. We present a method of DNA, RNA, or ribonucleoprotein (RNP) solution microinjection into the oviduct lumen and in vivo electroporation to target mucosal epithelial cells in restricted regions along the oviduct. There are several advantages of this method for cancer modeling, such as 1) high adaptability in targeting the area/tissue/organ and region of electroporation, 2) high flexibility in targeted cell types (cellular pliancy) when used in combination with specific promoters for Cas9 expression, 3) high flexibility in the number of electroporated cells (relatively low frequency), 4) no specific mouse line is required (immunocompetent disease modeling), 5) high flexibility in gene mutation combination, and 6) possibility of tracking electroporated cells when used in combination with a Cre reporter line. Thus, this cost-effective method recapitulates human cancer initiation.

Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation

This protocol describes microinjection and in vivo electroporation for regionally restricted CRISPR-mediated genome editing in the mouse oviduct.

מודלים של עכברים מהונדסים בגנום סומטי המשתמשים במיקרו-הזרקה ובאלקטרופורציה של Vivo

Germline genetically engineered mouse models (G-GEMMs) have provided valuable insight into in vivo gene function in development, homeostasis, and disease. ...

Cancer Research

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (&#62;1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

The present protocol describes zygote microinjection of CRISPR-Cas9 and donor DNA to efficiently produce gene cassette knock-in and floxed mice.

מיקרו-הזרקה של זיגוטה ליצירת קלטות גנטיות ואללים של פלוקס בעכברים

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily ...

<meta charset="utf8"></head><body>הליך הזרקת זרע עגול Murine לחקר התפתחות עוברית

Mouse Round Spermatid Injection

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of round spermatid injection (ROSI), oocytes can be successfully fertilized and developed into viable fetuses. In a groundbreaking milestone achieved in 1995, the first mouse fetus was born through ROSI technology. ROSI has since emerged as a pivotal tool for unraveling the intricate mechanisms governing embryonic development and holds significant potential in various applications, including the acceleration of mouse generation and the production of genetically modified mice. In 1996, a milestone was reached when the first human fetus was born through ROSI technology. However, the clinical applications of this method have shown a fluctuating pattern of success and failure. To date, ROSI technology has not found widespread application in clinical practice, primarily due to its low birth efficiency and insufficient validation of fetal safety. This article provides a comprehensive account of the precise methods of performing ROSI in mice, aiming to shed new light on basic research and its potential clinical applications.

<meta charset="utf8"></head><body>הזרקת זרע עגולה לעכבר

Here, we introduce a method for performing round spermatid injection (ROSI) in mice, a technique with promising clinical applications and utility for investigating the mechanisms underlying embryonic development.

הזרקת זרע עגולה לעכבר

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

הביצית Microinjection עכבר, הבשלה ו Ploidy הערכה

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

הדור של עכברים השתנה גנטית דרך Microinjection של ביציות

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השימוש של עוברים הקפאת-הפשפה לייצור יעילות גבוהה של עכברים ששונו גנטית

מודלים של עכברים מהונדסים בגנום סומטי המשתמשים במיקרו-הזרקה ובאלקטרופורציה של Vivo

מיקרו-הזרקה של זיגוטה ליצירת קלטות גנטיות ואללים של פלוקס בעכברים

הזרקת זרע עגולה לעכבר

הזרקת זרע עגולה לעכבר

הזרקת זרע עגולה לעכבר

הביצית Microinjection עכבר, הבשלה ו Ploidy הערכה