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A rapid protocol for integrating extrachromosomal arrays with high transmission rate into the C. elegans genome.
一种将具有高传输率的染色体外阵列整合到秀丽隐杆线虫基因组中的快速方案。

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Journal of Visualized Experiments : Jove, 09 Dec 2013, (82):e50773
可视化实验杂志 ： Jove， 09 Dec 2013， （82）：e50773
https://doi.org/10.3791/50773 PMID: 24379027 PMCID: PMC4396716
https://doi.org/10.3791/50773 PMID：24379027 PMCID：PMC4396716

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Abstract  抽象

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that exhibit partial and variable transmission rates of extrachromosomal arrays to the next generation. In addition, transgenic animals are mosaic and express the transgene in a variable number of cells. Extrachromosomal arrays can be integrated into the C. elegans genome using UV irradiation to establish nonmosaic transgenic strains with 100% transmission rate of the transgene. To that extent, F1 progenies of UV irradiated transgenic animals are screened for animals carrying a heterozygous integration of the transgene, which leads to a 75% Mendelian transmission rate to the F2 progeny. One of the challenges of this method is to distinguish between the percentage of transgene transmission in a population before (X% transgenic animals) and after integration (≥75% transgenic F2 animals). Thus, this method requires choosing a nonintegrated transgenic line with a percentage of transgenic animals that is significantly lower than the Mendelian segregation of 75%. Consequently, nonintegrated transgenic lines with an extrachromosomal array transmission rate to the next generation ≤60% are usually preferred for integration, and transgene integration in highly transmitting strains is difficult. Here we show that the efficiency of extrachromosomal arrays integration into the genome is increased when using highly transmitting transgenic lines (≥80%). The described protocol allows for easy selection of several independent lines with homozygous transgene integration into the genome after UV irradiation of transgenic worms exhibiting a high rate of extrachromosomal array transmission. Furthermore, this method is quite fast and low material consuming. The possibility of rapidly generating different lines that express a particular integrated transgene is of great interest for studies focusing on gene expression pattern and regulation, protein localization, and overexpression, as well as for the development of subcellular markers.
将 DNA 显微注射到秀丽隐杆线虫合胞体性腺的细胞质中是用于建立转基因系的主要技术，这些转基因系表现出染色体外阵列到下一代的部分和可变传递速率。此外，转基因动物是镶嵌的，并在不同数量的细胞中表达转基因。使用紫外线照射，可以将染色体外阵列整合到秀丽隐杆线虫基因组中，以建立具有100%转基因传播率的非嵌合转基因菌株。在这种程度上，对紫外线照射转基因动物的 F1 后代进行筛选，以筛选携带转基因杂合整合的动物，这导致 75% 的孟德尔传播率到 F2 后代。这种方法的挑战之一是区分整合前（X% 转基因动物）和整合后（≥75% 转基因 F2 动物）在种群中转基因传播的百分比。因此，这种方法需要选择转基因动物百分比明显低于孟德尔分离 75% 的非整合转基因系。因此，具有染色体外阵列向下一代传输率≤60%的非整合转基因系通常首选，并且很难在高传递菌株中进行转基因整合。在这里，我们表明，当使用高透射转基因系（≥80%）时，染色体外阵列整合到基因组中的效率会提高。所描述的方案允许在对表现出高染色体外阵列传输率的转基因蠕虫进行紫外照射后，轻松选择几个具有纯合转基因整合到基因组中的独立品系。 此外，这种方法速度快，材料消耗低。快速生成表达特定整合转基因的不同品系的可能性对于专注于基因表达模式和调控、蛋白质定位和过表达以及亚细胞标记物开发的研究非常感兴趣。

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J Vis Exp. 2013; (82): 50773.
J Vis Exp. 2013年;(82): 50773.
Published online 2013 Dec 9.
2013年12月9日在线发布。 https://doi.org/10.3791/50773
PMCID: PMC4396716 PMCID：PMC4396716
PMID: 24379027 PMID：24379027

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome
一种将具有高传输率的染色体外阵列整合到秀丽隐杆线虫基因组中的快速方案

Marie-Christine Mariol, 1 , 2 Ludivine Walter, 1 , 2 Stéphanie Bellemin, 1 , 2 and Kathrin Gieseler 1 , 2
玛丽-克里斯汀·马里奥尔、 1 , 2 卢迪万·沃尔特、 1 , 2 斯蒂芬妮·贝勒明和 1 , 2 凯瑟琳·吉塞勒 1 , 2
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Abstract 抽象

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that exhibit partial and variable transmission rates of extrachromosomal arrays to the next generation. In addition, transgenic animals are mosaic and express the transgene in a variable number of cells. Extrachromosomal arrays can be integrated into the C. elegans genome using UV irradiation to establish nonmosaic transgenic strains with 100% transmission rate of the transgene. To that extent, F1 progenies of UV irradiated transgenic animals are screened for animals carrying a heterozygous integration of the transgene, which leads to a 75% Mendelian transmission rate to the F2 progeny. One of the challenges of this method is to distinguish between the percentage of transgene transmission in a population before (X% transgenic animals) and after integration (≥75% transgenic F2 animals). Thus, this method requires choosing a nonintegrated transgenic line with a percentage of transgenic animals that is significantly lower than the Mendelian segregation of 75%. Consequently, nonintegrated transgenic lines with an extrachromosomal array transmission rate to the next generation ≤60% are usually preferred for integration, and transgene integration in highly transmitting strains is difficult. Here we show that the efficiency of extrachromosomal arrays integration into the genome is increased when using highly transmitting transgenic lines (≥80%). The described protocol allows for easy selection of several independent lines with homozygous transgene integration into the genome after UV irradiation of transgenic worms exhibiting a high rate of extrachromosomal array transmission. Furthermore, this method is quite fast and low material consuming. The possibility of rapidly generating different lines that express a particular integrated transgene is of great interest for studies focusing on gene expression pattern and regulation, protein localization, and overexpression, as well as for the development of subcellular markers.
将 DNA 显微注射到秀丽隐杆线虫合胞体性腺的细胞质中是用于建立转基因系的主要技术，这些转基因系表现出染色体外阵列到下一代的部分和可变传递速率。此外，转基因动物是镶嵌的，并在不同数量的细胞中表达转基因。使用紫外线照射，可以将染色体外阵列整合到秀丽隐杆线虫基因组中，以建立具有100%转基因传播率的非嵌合转基因菌株。在这种程度上，对紫外线照射转基因动物的 F1 后代进行筛选，以筛选携带转基因杂合整合的动物，这导致 75% 的孟德尔传播率到 F2 后代。这种方法的挑战之一是区分整合前（X% 转基因动物）和整合后（≥75% 转基因 F2 动物）在种群中转基因传播的百分比。因此，这种方法需要选择转基因动物百分比明显低于孟德尔分离 75% 的非整合转基因系。因此，具有染色体外阵列向下一代传输率≤60%的非整合转基因系通常首选，并且很难在高传递菌株中进行转基因整合。在这里，我们表明，当使用高透射转基因系（≥80%）时，染色体外阵列整合到基因组中的效率会提高。所描述的方案允许在对表现出高染色体外阵列传输率的转基因蠕虫进行紫外照射后，轻松选择几个具有纯合转基因整合到基因组中的独立品系。 此外，这种方法速度快，材料消耗低。快速生成表达特定整合转基因的不同品系的可能性对于专注于基因表达模式和调控、蛋白质定位和过表达以及亚细胞标记物开发的研究非常感兴趣。

Keywords:  关键字：Basic Protocol, Issue 82, Caenorhabditis elegans, UV-mediated transgene integration, transgenic worms, irradiation, extrachromosomal, fluorescent
基本方案，第 82 期，秀丽隐杆线虫，紫外线介导的转基因整合，转基因蠕虫，辐照，染色体外，荧光
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Introduction 介绍

Transgenes are extensively used in C. elegans for a large range of applications. Transgenic strains are typically generated by DNA injection into the syncytial hermaphrodite gonad1,2. The DNA carrying the transgene of interest is coinjected along with plasmids encoding coinjection markers1,3,4. Coinjection markers are established transgenes leading either to the expression of a fluorescent protein or to a specific behavioral or morphological phenotype. The injected DNAs rearrange to form multi copy extrachromosomal arrays; thus the transgene of interest and the coinjection marker are transmitted together5,4. Transgenic lines are selected in the F2 generation of injected animals by following the phenotype induced by the expression of the coinjection marker (fluorescence or specific phenotype)1. Transgenic lines exhibit partial and variable transmission rates of the extrachromosomal arrays to the next generation. Depending on the strain, 10-100% (100% being a rare event) of the animals inherit extrachromosomal arrays. In addition, transgenic animals are mosaic and express the transgene in a variable number of cells. This mosaicism is likely to be correlated to the transmission rate to the next generation. Indeed, in both germ line and somatic cells the transmission rate depends on the segregation of the extrachromosomal arrays during cell divisions. One way to avoid this issue is to integrate the transgene into the genome. Classically, the integration of a transgene into a chromosome relies on irradiation (ultraviolet or gamma) of transgenic worms carrying extrachromosomal arrays3. Briefly, 50-100 hermaphrodite animals are irradiated at the L4 larval stage. In the F1 generation 200-800 transgenic animals are selected and individually cultured onto plates. According to Mendelian segregation, transgenic F1 animals carrying a heterozygous integration of the transgene into the genome would produce 1/2 of transgenic descendants that are heterozygous for the integrated array, 1/4 of transgenic descendants being homozygous for the integrated array, and 1/4 of descendants that have not integrated the array (Figure 1). Thus, plates containing the F2 generation are visually screened for ≥75% transgenic animals as observed by the expression of the coinjection marker. About 1/3 of the F2 animals selected for being integrant candidates are assumed to be homozygous for the integrated array and to produce 100% of homozygous F3 animals. Hence, from each selected plate, three to eight transgenic F2 animals are individually cultured and plates with 100% of transgenic descendants are selected. Next, eight F3 animals are individually cultured to confirm the 100% inheritance of the transgene.
转基因在秀丽隐杆线虫中被广泛用于广泛的应用。转基因菌株通常通过将 DNA 注射到合胞体雌雄同体性腺中产生 1,2 。携带目的转基因的DNA与编码共注射标记的质粒一起注射 1,3,4 。共注射标记物是已建立的转基因，可导致荧光蛋白的表达或特定的行为或形态表型。注射的DNA重新排列以形成多拷贝染色体外阵列;因此，目的转基因和共注射标记一起 5,4 传递。在注射动物的F2代中，通过遵循共注射标记物（荧光或特异性表型） 1 的表达诱导的表型来选择转基因系。转基因系表现出染色体外阵列到下一代的部分和可变的传递率。根据菌株的不同，10-100%（100%是罕见事件）的动物遗传染色体外阵列。此外，转基因动物是镶嵌的，并在不同数量的细胞中表达转基因。这种嵌合体很可能与下一代的传播率有关。事实上，在生殖系和体细胞中，传输速率取决于细胞分裂过程中染色体外阵列的分离。避免这个问题的一种方法是将转基因整合到基因组中。传统上，转基因整合到染色体中依赖于携带染色体外阵列的转基因蠕虫的照射（紫外线或伽马射线 3 ）。简而言之，50-100 只雌雄同体的动物在 L4 幼虫阶段接受照射。 在 F1 代中，选择 200-800 只转基因动物并单独培养到平板上。根据孟德尔分离，携带转基因杂合整合到基因组中的转基因 F1 动物将产生 1/2 的转基因后代，这些后代对整合阵列是杂合的，1/4 的转基因后代对整合阵列是纯合的，1/4 的后代没有整合阵列（图 1）。因此，通过共注射标记物的表达观察到，对含有 F2 代的平板进行目视筛选 ≥75% 转基因动物。大约 1/3 的 F2 动物被选为整合候选物，假设它们是整合阵列的纯合子，并产生 100% 的纯合 F3 动物。因此，从每个选定的培养皿中，单独培养三到八只转基因F2动物，并选择具有100%转基因后代的培养皿。接下来，对8只F3动物进行单独培养，以确认转基因的100%遗传。

The main disadvantages of this method are that

  1. it requires a visual screen of several hundred F1 plates for ≥75% transgenic F2 progeny, which is time consuming due to the variable and unpredictable percentage of transmission of nonintegrated extrachromosomal arrays. Alternatively, to avoid the screening of F1 plates several subsequent starvation steps can be performed after irradiation of P0 animals; followed by picking 100 transgenic animals and screening for 100% transgenic progeny3.

  2. it is not adapted for strains exhibiting a percentage of extrachromosomal array transmission higher than 60% as this percentage is difficult to distinguish this percentage from 75%.

  3. as only strains with a relatively low transmission rate can be used, a high number of irradiated transgenic P0 animals is necessary as well as the observation of numerous F1 and F2 animals, which is highly time consuming and requires a considerable number of plates.

The irradiation of worms presumably induces double-strand breaks in the DNA and integration of extrachromosomal arrays into chromosomes occurs during DNA repair. Thus, it is likely that chances to obtain successful irradiation mediated integration are directly proportional to the number of germ line cells carrying an extrachromosomal array. Strains with a high transmission rate may contain more transgenic DNA arrays than strains with a low transmission rate as suggested in2, and exhibit a higher number of germ line cells carrying extrachromosomal arrays. Thus, we reasoned that in highly transmitting lines, transgenes might be easier to integrate than in lines with a low transmission rate. However, the standard protocol excludes the possibility of integrating transgenes with high transmission frequency as the percentage of transgenic animals with a nonintegrated extrachromosomal array can hardly be distinguished from that of the progeny of a F1 animal carrying a heterozygous integration of the transgene into the genome (Figure 1).

Here we show that for a particular transgene, the number of integrated lines recovered after irradiation with ultraviolet (UV) light is dependent on the initial percentage of transmission of the nonintegrated array. We present an improved protocol for UV irradiation-mediated transgene integration that is particularly relevant to lines exhibiting a high rate of transgene array transmission.

Protocol

1. Culturing Transgenic Nematodes Prior to Integration

Transgenic lines can be generated as described in1. These lines usually contain a coinjection marker plasmid leading either to the expression of a fluorescent protein or to a specific behavioral or morphological phenotype at a given development stage1,3. This integration protocol can be applied to strains expressing fluorescent or nonfluorescent coinjection markers. In the example to follow, a strain with a fluorescent coinjection marker was used.

  1. Prepare culture plates:

    1. Prepare about 500 60 mm large Petri dishes poured aseptically with 10 ml each of Nematode Growth Medium (NGM)6,7.

    2. Seed each plate with 0.3 ml of saturated Escherichia coli OP50 culture following standard procedure6,7.

  2. Evaluate the transmission rate of the transgenic line to be integrated:

    1. For each transgenic line, pick ten fluorescent gravid adults onto ten separate culture plates under a fluorescent stereoscope using a worm pick as described in7.

    2. Culture the animals in a worm incubator at 15-25 °C (depending on the genetic background) to allow the worms to reproduce.

    3. Observe progeny each day to evaluate at which developmental stage the coinjection marker is expressed and can be best observed. The expression of most of the currently used coinjection markers is observed at the L4 larval stage or at adulthood.

    4. Evaluate the transmission rate of the progeny by determining the percentage of fluorescent progeny by observation under a fluorescent stereoscope.

  3. Choose a transgenic line for transgene integration:

    1. Select the transgenic line with the highest transmission rate as possible (ideally ≥80%).

  4. Obtain a population of transgenic animals synchronized at the L4 larval stage for integration:

    1. Pick 30 fluorescent gravid adults onto five culture plates (six animals by plate).

    2. Let the worms lay eggs for 3-4 hr at 15 °C.

    3. Eliminate the adults from the plates after checking for the presence of at least 30 eggs by plate.

    4. Culture the animals in a worm incubator at 15-25 °C until the progeny reaches the L4 larval stage (96 hr at 15 °C or 36 hr at 25 °C).

2. UV Irradiation and Recovery of Transgenic Worms

  1. Worm irradiation:

    1. Pick 30-100 fluorescent transgenic L4 animals onto separate culture plates (15-20 animals by plate).

    2. Place the plates, with the lids removed, in a UV cross-linker. Irradiate the worms at 0.012 J/cm2. Note that strains are more or less sensitive to UV and that results can be variable depending on the UV irradiator used. A dose range between 0.010-0.015 J/cm2 can be tested.

  2. Worm recovery after irradiation:

    1. Place plates with irradiated worms overnight at 15 °C for recovery.

    2. Check for the number animals that are alive. In our hands, a survival rate of around 80-90% is adapted for an efficient irradiation.

    3. Grow the irradiated animals at 15-25 °C until the progeny has reached the developmental stage allowing for the observation of the coinjection marker expression, as established in step 1.2.

3. Isolation of Integrated Transgenic Lines

  1. Selection of F1 animals:

    1. Single 150-200 fluorescent F1 animals onto separate culture plates. For highly transmitting lines (≥80%), 100 F1 animals are enough.

    2. Keep these F1 plates at 15-25 °C until the progeny reaches an appropriate stage for the observation of fluorescent F2 animals.

    3. Discard all F1 plates exhibiting either i) no progeny, indicating that the F1 animal was sterile or died, or ii) no or only few fluorescent F2 animals, indicating that the F1 animal did not transmit the transgene at the expected rate. This step is quick and represents a real advantage when compared to the standard protocol that requires an estimation of the percentage of fluorescent F2 worms in order to select F1 plates exhibiting ≥75% fluorescent progeny (Table 1).

  2. Selection of F2 animals:

    1. Single four fluorescent transgenic F2 animals from each selected F1 plate. When using fluorescent transgenes (for example mCherry8, gfp9, or dsRed10), pick F2 worms with a high level of fluorescence as this could indicate that these animals are homozygous for the integrated array. Note that when picking F2 animals it is critical not to carry along eggs or larvae, in order to avoid false negatives in the next step.

    2. Grow F2 animals at 15-25 °C.

  3. Isolation and validation of integrated transgenic strains:

    1. Screen F2 plates for 100% fluorescent transgenic F3 worms. The screen is quite rapid as the presence of a single nonfluorescent worm indicates that the plate should be thrown away. A F2 animal homozygous for the integrated transgene will give 100% homozygous fluorescent progeny.

    2. Single eight fluorescent F3 animals from selected plates to confirm the 100% inheritance of the transgene. In theory, picking one animal is enough at this step. However, picking eight fluorescent F3 animals may be necessary to avoid selecting unfertile or unhealthy animals as irradiation can randomly induce mutations that affect genes important for worm physiology.

    3. If possible keep several independent integrated transgenic strains, i.e. strains recovered from different F1 animals.

Representative Results

Integrations of transgenes in two different lines transmitting extrachromosomal arrays at about the same frequency (50-60%) were performed using the standard and the improved protocols (Table 1). The standard protocol allowed the recovery of three independent integrated lines after UV irradiation of 91 transgenic L4 animals, while the improved protocol allowed the recovery of one integrated line after UV irradiation of 30 transgenic L4 animals. This suggests that the efficiency of integration is similar for both protocols (one integrated line per 30 irradiated L4 animals). However, we cannot rule out the possibility that one protocol could be more efficient than the other when compared for the integration of the same transgene. The main difference between the protocols is that with the improved method all F1 plates exhibiting fluorescent progeny were selected regardless of the percentage of fluorescent F2 animals, while with the standard method a visual screen of F1 plates was performed and only plates with ≥75% transgenic F2 progeny were selected. This screening step is highly time consuming, in particular if the initial rate of transmission is close to 75%. In the improved protocol, it is sufficient to single four F2 worms from each F1 plate carrying transgenic F2 animals and to screen for plates with 100% transgenic progeny (F3), which can be performed quickly. In addition, the improved protocol allows for a decrease in the number of analyzed F2 and F3 animals when compared to the standard protocol. Altogether, the time and the number of plates necessary per integrated line are optimized in the improved protocol.

Next, we tested the hypothesis that a higher transgene transmission rate in a nonintegrated line renders easier the integration of the transgene into the genome. One particular transgene was integrated in lines transmitting at different rates using the improved protocol (Table 2). Gonads of wild type worms were injected with 10 ng/µl of a plasmid (pKG45) carrying the Pdyc-1::dyc-1::gfp transgene and with 30 ng/µl of the coinjection marker plasmid carrying Pmyo-2::dsRed10. Several transgenic strains were recovered and three of them that transmitted at different levels (10-15%, 50-60%, and >80%, respectively) were selected. The transgene was integrated in each strain using the improved protocol. The protocol was performed under the same condition for each strain, except that a higher number of transgenic L4 animals were irradiated for the line with the lowest transmitting rate (10-15%). For the highest transmitting line (>80%) three integrated lines were recovered from only 90 selected F1 animals, while for the transgenic line transmitting at 50-60%, one integrated line was recovered from 110 selected F1 animals. For the line with the lowest transmission rate, we could not recover a line carrying the integrated transgene, although 524 F1 animals were selected. Furthermore, the number of plates required per integrated line was three times lower with the highest transmitting line than with the line transmitting at 50-60% (Table 2).

An external file that holds a picture, illustration, etc. Object name is jove-82-50773-0.jpg

Figure 1. Transgene transmission in the progeny of transgenic worms with a nonintegrated or a heterozygous integrated transgene.A) Without integration, a transgenic animal will transmit the transgene at a frequency of X%, B) If integration of the transgene into a chromosome occurs; the F1 is most likely heterozygous for the integration and may carry or not the extrachromosomal array (as indicated by the question mark). The F2 progeny will be composed of at least 75% of fluorescent worms due to the Mendelian segregation. Worms without integrated transgene can be fluorescent, if they have retained the nonintegrated array (left column of Panel B). Click here to view larger image.

An external file that holds a picture, illustration, etc. Object name is jove-82-50773-1.jpg

Table 1. Comparison of the standard and improved protocols for extrachromosomal arrays integration into the C. elegans genome. Two nonintegrated transgenes transmitting at about the same frequency were integrated using the standard or the improved protocol. The main difference lies in the fact that in the standard protocol a screen of the progeny on all F1 plates is performed to estimate the percentage of transgenic F2 worms, which should be ≥ 75%. This step takes a long time, particularly if the initial rate of transmission is close to 75%. In the improved protocol, it is sufficient to single four F2 worms from each F1 plate carrying transgenic F2 animals and to screen for 100% of transgenic worms in the progeny (F3), which can be performed quickly. The standard protocol is adapted from3.

An external file that holds a picture, illustration, etc. Object name is jove-82-50773-2.jpg

Table 2. Efficiency of transgene integration into the genome depends on the initial percentage of transgene transmission. Different nonintegrated lines, which transmitted the same transgene (Pdyc-1::dyc-1::gfp) at different rates (10-15%, 50-60%, and >80%, respectively) were used. Worms from each line were irradiated at the same UV dose (0.012 J/cm2) and the F3 generation screened for homozygous integration. The improved integration protocol applied to the highest transmitting line (>80%) recovered three integrated lines for only 74 selected F2 animals. The data presented with the line transmitting at 50-60% are those presented in Table 1 with the improved protocol.

Discussion

The protocol described here provides an efficient method to integrate extrachromosomal transgenic arrays into the genome of C. elegans. Using different nonintegrated lines, which transmitted the same transgene Pdyc-1::dyc-1::gfp (pKG45) at different rates (10-15%, 50-60%, and >80%), we showed that the number of integrated strains recovered after UV irradiation is dependent of the initial level of transmission of the nonintegrated line (Table 2). Therefore, transgenes are easier to integrate in lines exhibiting high transmission rate.

Integrated transgenes are of interest for a wide range of purposes including studies related to gene regulation and gene expression pattern. Integrated transgenes are also useful tools to establish fluorescent subcellular markers. When integrating transgenes it can be crucial to isolate several independent lines. Several studies require introducing the transgene in different genetic backgrounds. However, the transgene could be inserted on a chromosome near to the gene of interest or create a mutation that may lead to an undesired phenotype in a given genetic background. It is therefore valuable to generate several independent integrated lines with an easy and rapid method.

Integration of extrachromosomal arrays, which are generally composed of multiple copies of the transgenes, allows for the overexpression of the respective transgenes. Strains that overexpress a given transgene are useful tools to study i) the subcellular localization of proteins, which exhibit a low endogenous expression level, ii) the effect of the overexpression of a protein on worm physiology, or iii) the effect of the overexpression of a protein in a particular tissue11. Several applications require however the use of low or single copy integrated transgenes that can be generated with alternative methods such as coinjection of the transgene of interest along with single stranded oligos2, bombardment12, Mos1 transposon mediated Single Copy gene Insertion (MosSCI)13 or Cas-9-triggered homologous recombination14. The irradiation of worms with UV induces chromosomal breaks and thereby mutations. To limit this undesirable effect, doses between 0.010 and 0.015 J/cm2 may be used, which allows recovering of about 80-90% of healthy and fertile animals. Effects on worm physiology of the dose delivered depend on the distance between the UV lamp and the animals, which may vary between different UV cross linkers. Similar doses may lead to experimental variations if the conditions of irradiation are not carefully controlled. Thus, it is important for each lab to determine the most suited UV dose for its particular settings and strains. Irradiated worms presenting fertility and/or survival rates close to that of nonirradiated worms indicate that the UV dose used was too low. Conversely, a high mortality rate (more than 40% of the animals) after UV irradiation indicates that the UV irradiation dose is potentially too high. Importantly, we noticed that worms placed at 15 °C for 24 hr after irradiation at 15° were healthier and gave greater progeny than worms directly grown at 23 °C. Thus, a recovery step at 15 °C after irradiation as presented here is critical for the survival and health of the animals. Alternatively, gamma irradiation can be used instead of UV irradiation to integrate extrachromosomal arrays in the C. elegans genome. This method requires however more sophisticated equipment3.

Several reasons could be considered in case the improved protocol presented here fails to recover integrated transgenic lines. The number of irradiated P0 animals or the number of selected F1 or F2 animals may be insufficient to recover integrated lines. It is also possible that nontransgenic eggs or larvae were carried along during transgenic F2 animals picking, thus leading to false negatives in the F3 progeny. Finally, some transgenes may be toxic, when expressed at high levels. Another pitfall of the integration procedure can come from toxicity of the transgene, especially when a strong promoter is used to express the gene of interest. The toxicity of a transgene can be masked by its mosaic expression in nonintegrated lines but revealed in integrated lines provoking the death of the animals. In this case aforementioned alternative methods could be used to produce lines with low copy integrated transgenes.

The presented protocol was optimized for the integration of extrachromosomal arrays in highly transmitting lines (>80%) that have higher chances to integrate a transgene into their genome. However, for particular transgenes, highly transmitting lines may not be available after worm transformation. When using lines transmitting the transgene at a rate of ≤50%, the standard method that includes an extra screening step of F1 plates may be more appropriate for successful transgene integration.

Disclosures

The authors declare that they have no competing financial interest.

Acknowledgments

Authors would like to acknowledge Claire Lecroisey and Laurent Ségalat for generating nonintegrated Pdyc-1::dyc-1::gfp; Pmyo-2::dsRed and Pmyo-3::gfp transgenic strains. We thank Andy Fire for the gift of the Pmyo-2::dsRed plasmid and Pamela Hoppe for the gift of the Pmyo-3::gfp plasmid. This work was founded by the French National Center of Scientific Research (CNRS) and the Claude Bernard University of Lyon.

References

  • Berkowitz LA, Knight AL, Caldwell GA, Caldwell KA. Generation of stable transgenic C. elegans using microinjection. J. Vis. Exp. 2008. p. e833. [Europe PMC free article] [Abstract]
  • Mello C, Kramer JM, Stinchlomb D, Ambros V. Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 1991;10(12):3959–3970. [Europe PMC free article] [Abstract] [Google Scholar]
  • Evans TC, editor. WormBook. The C. elegans Research Community, WormBook; 2006. Transformation and microinjection. [Google Scholar]
  • Hobert O. PCR Fusion-Based Approach to Create Reporter Gene Constructs for Expression Analysis in Transgenic C. elegans. BioTechniques. 2002;32:728–730. [Abstract] [Google Scholar]
  • Stinchcomb DT, Shaw JE, Carr SH, Hirsh D. Extrachromosomal DNA transformation of Caenorhabditis elegans. Mol. Cell. Biol. 1985;5:3484–3496. [Europe PMC free article] [Abstract] [Google Scholar]
  • Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77:71–94. [Europe PMC free article] [Abstract] [Google Scholar]
  • Chaudhuri J, Parihar M, Pires-daSilva A. An Introduction to Worm Lab: from Culturing Worms to Mutagenesis. J. Vis. Exp. 2011. p. e2293. [Europe PMC free article] [Abstract]
  • Merritt C, Rasoloson D, Gallo C, Voronina E, Ko D, Seydoux G. A modular vector kit for transgenesis in C. elegans. International Worm Meeting. 2007.
  • Chalfie M, Tu Y, Euskirchen G, Ward W, Green Prasher D. Fluorescent protein as a marker for gene expression. Science. 1994;263:802–805. [Abstract] [Google Scholar]
  • Fleenor J, Fire A. Discosoma sp. Red Fluorescent Protein (dsRED) as a reporter in C. elegans. Worm Breeder's Gazette. 2000;16(3):16. [Google Scholar]
  • Lecroisey C, et al. ZYX-1, the unique zyxin protein of Caenorhabditis elegans, is involved in dystrophin-dependent muscle degeneration. Mol. Biol. Cell. 2013. [Europe PMC free article] [Abstract]
  • Praitis V, Casey E, Collar D, Austin J. Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics. 2001;157(3):1217–1226. [Europe PMC free article] [Abstract] [Google Scholar]
  • Frøkjaer-Jensen C, et al. Single-copy insertion of transgenes in Caenorhabditis elegans. Nat. Genet. 2008;40(11):1375–1383. [Europe PMC free article] [Abstract] [Google Scholar]
  • Dickinson DJ, Ward JD, Reiner DJ, Goldstein B. Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat. Methods. 2013;10(10):1028–1034. [Europe PMC free article] [Abstract] [Google Scholar]
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