Name: pregnancy and nursing management for embryo-transferred and genetically modified rabbits
Uploaded: 2024-12-13T14:50:00.000+00:00
Duration: 4 min 29 s
Description: With the advancement of scientific research, the demand for gene-edited rabbit models is increasing. However, there are limited pregnancy and feeding ...

This research was supported by the National Natural Science Foundation of China (Grant No. 82101937), the Guangdong Medical Science and Technology Research Fund Project, China (Grant No. B2024069), and the Guangzhou Science and Technology Plan Project, China (Grant No. SL2023A04J02229, Assignment No. 2024A04J4923).

All experimental protocols were approved by the Ethics Committee for Animal Experiments of the Guangdong Medical Laboratory Animal Center. The ethical review number for the MKRN3 gene-modified rabbit breeding involved in this study is B202210-6. The study adheres to procedures in compliance with ethical standards outlined in the Helsinki Declaration of 1975 (revised 1983). The New Zealand rabbits used in this study were obtained from the Guangdong Medical Laboratory Animal Center in China. The pregnancy and feeding process of genetically modified rabbits described in this article is a summary of the daily animal breeding practices in the author&#39;s laboratory. Details on the reagents and the equipment used are listed in the Table of Materials.
1. Pregnancy diagnosis
<ol>
	<li>On the 12th to 14th day after embryo transfer (choose one of the time points), confirm pregnancy through direct palpation. 
	NOTE: This step can be skipped. If palpation is too rough and forceful, it can lead to miscarriage in pregnant rabbits.
	<ol>
		<li>Hold the neck skin behind the rabbit&#39;s ears with one hand, let the rabbit&#39;s buttocks shrink forward, close to the abdominal cavity of the rabbit&#39;s buttocks, and then use the other hand to open the palm and gently palpate the back end of the abdomen.</li>
		<li>Press the abdomen to a certain degree. If a continuous string of smooth and elastic flesh balls, similar in size to fava beans, is palpated, it is considered pregnancy24.</li>
	</ol>
	</li>
	<li>If conditions permit, use ultrasound on the 15th day after embryo transfer to determine if the rabbit is pregnant. The pregnancy diagnosis of rabbits is based on the presence of small, strong echogenic points (gestational sacs) in the liquid dark zone.</li>
	<li>During the period of the 16th-18th days after embryo transfer, observe the rabbit twice a day in the morning and evening, for approximately 15 min each time. If the rabbit is observed to exhibit behaviors such as restlessness, rapid breathing, plucking hair to build nests, etc., during labor or estrus, it indicates that the female rabbit is pseudopregnant and has not truly conceived. 
	NOTE: If embryo transfer fails, female rabbits will have pseudopregnancy similar to parturient or oestrus on the 16th-18th days due to the decrease of luteal hormone.</li>
</ol>
2. Antenatal care
<ol>
	<li>Allow pregnant rabbits to eat freely during pregnancy and add fresh green feed once a week.</li>
	<li>On the morning or evening of the 25th day after embryo transfer, transfer the pregnant rabbits to the delivery cage.
	<ol>
		<li>Disinfect the delivery cage, animal incubator, paper scraps, or shavings in advance. 
		NOTE: Choose a rabbit delivery cage that meets international criteria in size. Ensure that the delivery cage has sufficient space for the mother rabbit to move. Choose an incubator that can resist mother rabbit nibble. Choose thick paper scraps with good water absorption. According to the requirements and suggestions of local animal facility management, select appropriate disinfection methods.</li>
		<li>Fix the incubator on one side of the delivery cage, and put towels, shavings, or paper scraps into the animal incubator for the pregnant rabbits to make a nest.</li>
		<li>Provide adequate feed and drinking water.</li>
		<li>Gently move the pregnant rabbits to the delivery cage.</li>
		<li>Pay attention to whether the pregnant rabbits have made a nest in the incubator every day. If not, add some paper scraps and towels outside the incubator on the morning of the 28th day of pregnancy. 
		NOTE: If the pregnant rabbits do not nest in the incubator, it will likely not give birth in the incubator. Put some towels and paper scraps outside the incubator to prevent newborn rabbits from losing temperature and dying outside the box.</li>
	</ol>
	</li>
</ol>
3. Parturition
NOTE: Day 0 refers to shortly after fertilization and prokaryotic formation. Rabbits are ovulation-stimulating animals that ovulate 10-12 h after mating. The single-cell stage embryos obtained on the day after mating are considered to be on the 0.5th day. If single-cell stage embryos are cultured in vitro for only 2-3 h and then transferred to surrogate rabbits, it is also considered to be the 0.5th day of pregnancy.
<ol>
	<li>Prenatal monitoring 
	NOTE: The gestation period of rabbits is 28-32 days. If a rabbit gives birth before the 30th day of pregnancy, the size of the fetus is usually relatively small, and there are rare cases of difficult labor. No excessive intervention is needed before the 30th day.
	<ol>
		<li>On the morning of the 30th day of pregnancy in rabbits, perform abdominal palpation to roughly determine the number, size, and activity of the fetus.
		<ol>
			<li>Hold the neck skin behind the rabbit&#39;s ears with one hand, let the rabbit&#39;s buttocks shrink forward, close to the abdominal cavity of the rabbit&#39;s buttocks, and then use the other hand to open the palm and gently palpate the back end of the abdomen. The force must be light. 
			NOTE: Pressing into the abdomen to a certain extent will palpate several egg-sized ellipsoids, which can feel the movement of the fetus.</li>
		</ol>
		</li>
		<li>If it is found that the number of fetuses conceived by pregnant rabbits is only 1-2, on the 30th day of pregnancy, inject 0.02 mg of cloprostenol intramuscularly at 5 p.m. to induce the rabbits&#39; delivery the next day. 
		NOTE: Pregnant rabbits have only 1-2 fetuses, which are prone to dystocia or delivery failure when mature.</li>
	</ol>
	</li>
	<li>Hasten parturition 
	NOTE: If the pregnant rabbit does not give birth on the morning of the 31st day of pregnancy, it can consider whether it needs to hasten parturition according to the following conditions.&#160;If there are more than 2 fetuses, no further action will be taken.
	<ol>
		<li>If the pregnant rabbit is only pregnant with 1-2 fetuses and has been injected with cloprostenol the day before, on the morning of the 31st day of pregnancy, inject 10 units of oxytocin into the muscles to induce labor.&#160;</li>
		<li>If the pregnant rabbit has signs of delivery (such as restlessness, rapid breathing, ear vascular dilation, plucking and making a nest, planning the ground, etc.), inject 10 units of oxytocin into the muscles to induce labor.</li>
		<li>If the pregnant rabbit has no signs of delivery and fetal movement is normal, continue to observe. If there is no delivery in the morning of the 32nd day of pregnancy, inject 10 units of oxytocin into the muscles to induce labor. 
		NOTE: Usually, the use of oxytocin can solve difficult labor. Generally, 5-10 min after oxytocin injection, the pregnant rabbits will start delivery. Oxytocin is effective for about half an hour and can be administered again after half an hour. It is not recommended to induce labor with oxytocin more than 3 times. If oxytocin is ineffective, have a veterinarian perform a cesarean section. Otherwise, the fetus will die in the uterus, soften, and be discharged from the vagina.</li>
	</ol>
	</li>
</ol>
4. Midwifery
<ol>
	<li>Arrange and prepare the incubators (Figure 1). 
	NOTE: Prepare in advance before the rabbit gives birth. The size of the incubator used in this study is 45 cm &#215; 30 cm &#215; 15 cm in length &#215; width &#215; height.
	<ol>
		<li>Add sterile absorbent padding. Corncob granules are recommended.</li>
		<li>Spread a towel as a mat covered with artificial down cotton. 
		NOTE: The material of artificial down cotton is baby-grade polyester fiber, which is non-toxic. Artificial down cotton is very suitable for newborn rabbits keeping warm. However, it cannot be used to make a nest for the mother rabbit, or it may be eaten into the stomach.</li>
	</ol>
	</li>
	<li>Quickly wipe the mucus on the newborn rabbits with a towel. Ensure they breathe smoothly, and then put them into the incubator. 
	NOTE: If the breathing of newborn rabbits stops, immediately press the chest for auxiliary breathing (25-35 times/min) to reduce the chest diameter by 25%-30% until spontaneous breathing is restored.</li>
	<li>After the delivery of pregnant rabbits, count the number of fetuses born. Stick information cards on the incubator: modified genes, number of newborn rabbits, date of birth, etc. 
	NOTE: The suitable environmental temperature for newborn rabbits is 30-32 &#176;C. Placing an appropriate amount of rabbit hair or artificial down cotton inside the incubator can achieve a good insulation effect. Rabbit house should be maintained at 16-25 &#176;C. Female rabbits usually have 6-8 nipples. If there are too many babies, some babies can be fed by other mother rabbits.</li>
</ol>
5. Postpartum care
<ol>
	<li>Baby rabbit care (Figure 2) 
	NOTE: After giving birth, let the mother rabbit rest for about 1 h before breastfeeding. Rabbits usually breastfeed 1-3 times a day.
	<ol>
		<li>Clean all nipples of the mother rabbit with alcohol cotton before the first feeding. Except for breastfeeding, keep mother rabbits and baby rabbits separately at other times.</li>
		<li>Gently massage each nipple and gently squeeze out the first drop of milk.</li>
		<li>Feeding of 1-2-day-old baby rabbits: Each time, place the mother rabbit on the mat in a lateral position or a prone position and place the sucking rabbits one by one to their nipples to suck milk (Figure 2A). 1-2 day-old rabbits need to be fed 2-3 times a day. 
		NOTE: Premature rabbits or low birth weight baby rabbits are prone to hypoglycemia. Feed 1 mL of 5% glucose and consume breast milk as soon as possible. If the baby rabbit is too weak to drink, then directly inject 1 mL of 5% glucose into the abdominal cavity.</li>
		<li>On the second day after birth, use a marker pen to number the back of each baby rabbit. Trim a small amount of tail tissue for genotyping.</li>
		<li>Feeding baby rabbits over 2 days old: When feeding, place the mother rabbit in an incubator and let it breastfeed on its own (Figure 2B). 2-9-day-old rabbits need to be fed 1-2 times a day, and over 9-day-old rabbits need to be fed once a day until weaning. Make sure the baby rabbits are full (Figure 2C). 
		NOTE: If the mother rabbit is unwilling to breastfeed, it can be fed some fresh vegetables to make it more willing to cooperate. If necessary, mother rabbits can be breastfed after being placed in a stable position. Gently squeeze out the first drop of milk from each nipple of the mother rabbit to alleviate breast swelling and pain, which is beneficial for the baby rabbits to suckle on the milk. If the above methods are ineffective, other lactating mother rabbits should be used for breastfeeding.</li>
		<li>During the first 4 days after birth, clean the perineum of the baby rabbit twice a day with wet cotton balls to stimulate the excretion of meconium and urine (Figure 2D).</li>
		<li>As the hair of the baby rabbit grows, remove some down cotton from the incubator every day.</li>
		<li>Change the towel once a day until the baby rabbit grows to the point where its entire body is covered in hair. 
		NOTE: Generally, the towel is no longer needed on the 8th day after birth.</li>
		<li>If baby rabbits (1-2 weeks old) experience diarrhea, immediately feed 3 drops of animal-grade gentamicin (concentration is 0.04 g/mL) twice a day for 5 consecutive days. 
		NOTE: Diarrhea in baby rabbits is usually caused by sucking milk from mother rabbits with mastitis, or by the high temperature and humidity of the environment. The mortality rate of baby rabbits with diarrhea is very high.</li>
		<li>After the young rabbit opens its eyes (about 2 weeks old), move it to a nursing cage with a towel.&#160;Add some green feed and daily diet. 
		NOTE: The suitable temperature for baby rabbits (2-3 weeks old) is 23-30 &#176;C.</li>
		<li>Separate the mother rabbit from the baby rabbits and only open the baffle once a day for breastfeeding. 
		NOTE: This can prevent the baby rabbits from biting the mother rabbit&#39;s nipple.</li>
	</ol>
	</li>
	<li>Postpartum care of the mother rabbit
	<ol>
		<li>During lactation, allow mother rabbits to drink and eat freely and feed them with appropriate green feed to ensure adequate nutrition.</li>
		<li>If the mother rabbit suffers from mastitis, stop breastfeeding immediately and administer appropriate veterinary antibiotics for treatment (cephalosporins are recommended). Feed the young rabbits to other lactating mother rabbits with similar delivery times.</li>
	</ol>
	</li>
</ol>
6. Ablactation
<ol>
	<li>Ensure that most young rabbits are weaned at 35 days of age. Weak rabbits are weaned at 40 days of age. 
	NOTE: The suitable temperature for young rabbits (3 weeks to 3 months old) is 20-25 &#176;C. The suitable temperature for young rabbits (3-6 months old) and adult rabbits is also 15-25 &#176;C.</li>
	<li>Consider whether to vaccinate conventional vaccines according to the experimental requirements after weaning.</li>
</ol>

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The authors have nothing to disclose.

The main steps in the pregnancy and nursing management of embryo-transferred and genetically modified rabbits include midwifery and early assisted breastfeeding. Difficult childbirth is a common issue in rabbits19. Prolonged delivery times often result in fetal ischemia, hypoxia, and death25. The appropriate use of oxytocin can effectively shorten delivery times and reduce instances of fetal dystocia26. Many primiparous mother rabbits fail to pluck hair to prepare nests, increasing the risk of temperature loss and mortality in newborn rabbits27,28. Typically, after an intramuscular injection of oxytocin, rabbits begin delivery within 5-10 min, which facilitates artificial delivery and improves the survival rate of newborns. Poor maternal behavior or inexperience often hinders the proper growth of rabbit pups. Assisted nursing management ensures that newborn rabbits receive adequate breastfeeding and are housed in comfortable incubators, significantly improving their survival29.
In this study, a simple incubator was utilized, employing artificial down cotton and towels for warmth. The optimal environmental temperature for newborn rabbits is 30-32 &#176;C30. If available, temperature and humidity control equipment can further enhance outcomes. In the late stages of pregnancy, female rabbits may exhibit symptoms such as accelerated breathing, respiratory rales, ear vasodilation, and reluctance to eat or move31. These symptoms resemble human pregnancy-induced hypertension and require immediate attention. In severe cases, maternal and fetal death may occur. Under such circumstances, pregnant rabbits should be housed in more spacious cages with paper scraps to encourage nest-making, promoted to exercise, and provided with fresh green feed. These measures generally alleviate adverse symptoms in pregnant rabbits. However, in critical cases, an intramuscular injection of 0.02 mg of cloprostenol is necessary to induce early pregnancy termination32.
This study establishes a pregnancy and nursing management protocol for embryo-transferred and genetically modified rabbits, significantly improving the breeding efficiency of genetically modified rabbits. Compared to traditional rabbit farm breeding methods, this approach requires more manpower and material resources and involves a more meticulous process. However, for standard laboratory settings, this method meets experimental needs and offers practical insights and skills.
When breeding rabbit models for specific genetic diseases, such as muscular atrophy rabbits or hairless rabbits, the weak congenital constitution of these models often results in their inability to compete with wild-type rabbits in the same litter for breast milk, leading to high mortality rates shortly after birth33,34. Assisted nursing management effectively mitigates these challenges, preventing the premature death of rabbit pups. Additionally, this protocol allows researchers to observe and determine the genotype of newborn rabbits promptly after birth, reducing concerns about female rabbits biting or eating their offspring. Overall, this protocol has promising applications in the generation of embryo-transferred and genetically modified rabbits.

Rabbits are a classic animal model for biomedical research and are increasingly becoming the preferred translational model to bridge the gap between rodent models and large animal models1,2. Compared with large animals, rabbits have moderate body size, small feeding space, low feeding cost, and convenient blood collection, which are conducive to repeated collection of research data and surgical operations. Rabbits are characterized by good reproductive ability, a short gestation period (28-32 days for New Zealand white rabbits), a large number of births, and fast growth3,4. Compared with rodents, rabbits are closer to humans in phylogenesis5. It is a cost-effective and practical experimental substitute for pigs and non-human primates6. Compared with other large and medium-sized gene editing animals (pigs, cattle, sheep, cats, dogs, monkeys), the preparation cost of gene editing rabbits is low, and the cycle is short. With the development of gene therapy7,8,9, stem cell therapy10,11,12,13, brain science research14, and other scientific fields1,15,16,17,18, the demand for nonrodent species in gene editing is increasing.
Gene editing rabbits were once considered to be one of the middle-sized gene editing animals that could realize industrial applications6. However, until today, gene-editing rabbits have not been able to achieve large-scale production. One of the main reasons is that the production and breeding of gene-editing rabbits are challenging, and the difficulty is much greater than that of gene-editing mice or rats. There are not many laboratories that can efficiently cultivate gene-editing rabbits. It is often difficult for beginners to successfully prepare gene editing rabbits, and they often encounter such unpleasant problems as low pregnancy rate19, abortion20, dystocia21, refusal of lactation by female rabbits22, and death of suckling rabbits23. However, there are few systematic data or literature guidelines on the pregnancy and feeding of gene-editing rabbits or embryo-transferred rabbits. Most of them are based on the experience of ordinary rabbit farms.
Therefore, this article summarizes the mature experience of pregnancy and nursing management of genetically modified rabbits from author&#39;s laboratory, and introduces a set of basic processes of embryo-transferred rabbits and genetically modified rabbits, including pregnancy diagnosis, antenatal care, midwifery, artificial assisted feeding, ablactation, as well as the rescue and nursing of weak baby rabbits. This process is specially established for breeding gene-editing rabbits or embryo-transferred rabbits. Compared with the traditional natural childbirth and nurturing method of rabbit farms, the pregnancy and nursing management model takes more time and energy, but it can greatly reduce the mortality of baby rabbits, and the effort is worth it.

<table><tbody><tr><td>Artificial down cotton</td><td>Yangzhou Anguang Textile Co., Ltd, China</td><td>5490-Jinyu</td><td></td></tr><tr><td>Cloprostenol Sodium Injection</td><td>Shanghai Quanyu Biotechnology (Zhumadian) Animal Pharmaceutical Co., Ltd, China</td><td>163232207</td><td></td></tr><tr><td>Corncob granules</td><td>Guangdong Provincial Medical Laboratory Animal Center, China</td><td>YUMIXIN</td><td></td></tr><tr><td>Electronic platform scale</td><td>Yongkang Runjin weighing instrument Co., Ltd, China</td><td>rj-09</td><td></td></tr><tr><td>Gentamicin</td><td>Shanxi Jinfukang Biological Pharmaceutical Co., Ltd, China</td><td>041531504</td><td></td></tr><tr><td>Glucose injection</td><td>Henan Kelun Pharmaceutical Co., Ltd, China</td><td>H41022251</td><td></td></tr><tr><td>Incubator</td><td>Foshan Chancheng Hualong Plastic Factory, China</td><td>AAA-2</td><td>For newborn rabbits</td></tr><tr><td>Incubator box&amp;nbsp;</td><td>Hebei mabao wire mesh products Co., Ltd., China</td><td>https://qr.1688.com/s/AJ1K7O3p</td><td>As a nest for the mother rabbits</td></tr><tr><td>Inslin Syringe</td><td>Becton,Dickinson and Company, USA</td><td>Ultra-Fine,328421</td><td></td></tr><tr><td>Oxytocin injection</td><td>Guangzhou Baiyunshan Mingxing Pharmaceutical Co., Ltd</td><td>H44025245</td><td></td></tr><tr><td>Penicillin injection</td><td>Guangdong Kangtaiyuan Animal Husbandry Co., Ltd, China</td><td>300012430</td><td></td></tr><tr><td>Rabbit delivery cage</td><td>Suzhou Suhang Technology Equipment Co., Ltd., Suzhou, China</td><td>RB42-8G</td><td></td></tr><tr><td>Towel</td><td>Zhejiang Jieliya Co., Ltd, China</td><td>W3290</td><td></td></tr></tbody></table>

pregnancy and nursing management for embryo-transferred and genetically modified rabbits

With the advancement of scientific research, the demand for gene-edited rabbit models is increasing. However, there are limited pregnancy and feeding management systems for gene-edited rabbits, leading to low survival rates among gene-edited rabbits prepared by many inexperienced researchers. Therefore, proper guidance is essential. This article summarizes the pregnancy and feeding practices for genetically modified rabbits developed in the author's laboratory and outlines a set of fundamental processes. These include pregnancy diagnosis, antenatal care, midwifery, assisted breastfeeding, weaning, and other procedures, along with the rescue and care of weak newborn rabbits. Compared to the traditional natural childbirth and nurturing methods used in rabbit farms, this approach involves more refined management, requiring additional time and effort but significantly increasing the survival rate of suckling rabbits. The methods described in this article are suitable for most laboratory breeding scenarios involving gene-edited or embryo-transferred rabbits and provide a straightforward and effective reference for other researchers.

This article describes a pregnancy and feeding management procedure for embryo-transferred and genetically modified rabbits. At the end of 2022, the author&#39;s laboratory initiated the development of a preparation platform for gene-modified rabbits. During this period, several common issues were encountered, including abortion, failure to deliver at term, dystocia, failure to pluck hair for nest preparation before delivery, the unwillingness of mother rabbits to breastfeed or incidents of cannibalism, insufficient breast milk due to large litter sizes, and baby rabbits failing to drink milk. These challenges significantly reduced the breeding efficiency of gene-modified rabbits.
By summarizing experiences and learning from these issues, a pregnancy and nursing management protocol for embryo-transferred and genetically modified rabbits was developed (Figure 3). This protocol has substantially improved the survival rate of newborn rabbits and reduced mortality during lactation (Table 1). For most cases, except for specific genetically modified disease models with severe postnatal genetic disease phenotypes leading to higher mortality rates, this approach has proven effective and acceptable.
Currently, the author&#39;s laboratory has transitioned from traditional natural childbirth and nurturing methods to the newly established pregnancy and nursing management model. To date, this method has facilitated the successful breeding of more than ten new varieties of genetically modified rabbits, resulting in the production of numerous offspring. Although this approach increases the workload, it is highly effective. Figure 4 illustrates a real-life example of breeding MKRN3 gene-modified rabbit offspring, showcasing the process from birth to weaning.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67793/67793fig01.jpg" /> 
Figure 1: Arrangement of baby rabbit incubators. Steps involved in preparing an incubator for baby rabbits: (1) Preparing the incubator; (2) Padding; (3) Spreading a towel; (4) Adding artificial down cotton; (5) Placing the baby rabbit inside; (6) Attaching the information card. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67793/67793fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67793/67793fig02.jpg" /> 
Figure 2: Assisted breastfeeding for baby rabbits. (A) Feeding of 1-2-day-old baby rabbits. (B) Feeding of baby rabbits over 2 days old. (C) Comparison of rabbit pups&#39; abdomens before and after feeding, showing noticeable bulging and visible white breast milk in the stomach (indicated by the red circle). (D) Excretion of meconium from the gastrointestinal tract within 3 days of birth (indicated by the red arrow). The perineum requires regular cleaning with a wet cotton ball (indicated by the yellow arrow). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67793/67793fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67793/67793fig03.jpg" /> 
Figure 3: Pregnancy and nursing management process for embryo-transferred and genetically modified rabbits. Steps in the management process: Pregnancy diagnosis; Antenatal care; Midwifery; Postpartum care; Ablactation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67793/67793fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67793/67793fig04.jpg" /> 
Figure 4: Growth and development of genetically modified rabbits from birth to ablactation. (A) Images of baby rabbits from 1-8 days old. (B) Fifteen-day-old baby rabbits, with the mother rabbit on the left and the baby rabbits on the right. (C) Thirty-five-day-old weaned young rabbits. (D) Growth curve of baby rabbits within the first three weeks after birth. Baby rabbits were from the same litter: ID1, ID2, and ID3 represent mkrn3 gene-modified rabbits, while ID4, ID5, and ID6 represent wild-type rabbits. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67793/67793fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Traditional natural childbirth and nurturing</td>
			<td>Pregnancy and nursing management</td>
		</tr>
		<tr>
			<td>The number of newborn rabbits (8 litters)</td>
			<td>68</td>
			<td>71</td>
		</tr>
		<tr>
			<td>The number of surviving newborn rabbits</td>
			<td>42</td>
			<td>66</td>
		</tr>
		<tr>
			<td>The number of surviving weaned rabbits</td>
			<td>29</td>
			<td>63</td>
		</tr>
		<tr>
			<td>Birth survival rate (= newborn rabbits / surviving newborn rabbits &#215; 100%)</td>
			<td>61.80%</td>
			<td>93.00%</td>
		</tr>
		<tr>
			<td>Weaning survival rate (= surviving weaned rabbits / newborn rabbits &#215; 100%)</td>
			<td>69.00%</td>
			<td>95.50%</td>
		</tr>
	</tbody>
</table>
Table 1: Survival of rabbit pups under different breeding management modes. A comparison of rabbit pup survival rates across various breeding management strategies.

This protocol describes a pregnancy and feeding management technique for embryo-transferred and genetically modified rabbits, aimed at reducing newborn rabbit mortality and enhancing the preparation efficiency of gene-edited rabbits.

Pregnancy and Nursing Management for Embryo-Transferred and Genetically Modified Rabbits

With the advancement of scientific research, the demand for gene-edited rabbit models is increasing. However, there are limited pregnancy and feeding ...

pregnancy-nursing-management-for-embryo-transferred-genetically

Nanyang Technological University

Research

JoVE Journal

Biology

209 Views.  Guangzhou Medical University. Dit protocol beschrijft een zwangerschaps- en voedingsmanagementtechniek voor embryo-overgeplaatste en genetisch gemodificeerde konijnen, gericht op het verminderen van de sterfte van pasgeboren konijnen en het verbeteren van de bereidingsefficiëntie van gen-bewerkte konijnen.

Video: Zwangerschaps- en borstvoedingsmanagement voor embryo-overgeplaatste en genetisch gemodificeerde konijnen

Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced, as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population ...

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.

In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

A Nature Editorial in 2003 asked the question  "Good-bye, flat biology?"  What does this question imply? In the past, many in vitro culture systems,  ...

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios &lt;0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (&lt; 5% events with cytoplasmic PI staining).

An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor&bull;hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR&bull;hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. 
The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR&bull;hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR&bull;hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid. 
Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6. 
In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR&bull;hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.

An in vitro method for preparing functional glucocorticoid receptor (GR)&#x2022;hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

Pregnancy and Nursing Management for Embryo-Transferred and Genetically Modified Rabbits

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