Gene insertion site and pattern experiments
Gene insertion site and pattern experiments
The study of insertion patterns and loci of exogenous genes in transgenic plants greatly contributes to the understanding of the mechanisms of integration, expression and stable inheritance of exogenous genes in plant nuclear genomes. This chapter introduces the standard procedures and methods for analyzing the insertion pattern and number of exogenous genes in transgenic cereal crops and forage grasses obtained by Agrobacterium tumefaciens-mediated transformation or direct transformation with exogenous DNA. The source of this experiment is "A Guide to Transgenic Technology and Field Identification of Wheat Crops" (H.D. Jones, P.R. Hewley, ed.). Hewley, eds.
Operation method
Gene insertion sites and patterns
Materials and Instruments
dCTP Move I. Number of transgene insertion sites For more product details, please visit Aladdin Scientific website.
Ethanol Sodium hypochlorite β-glucuronidase gene activity assay solution Ethidium bromide
Culture chamber MS medium
The number of insertion sites of the exogenous gene in the first generation ( T0 ) of transgenic plants is generally identified by genetic methods. Although genetic analysis can be performed in any generation, the second generation ( T1 ) obtained from self-crossing of transgenic plants, or from crosses with the wild type, is generally chosen for this purpose. The phenotypic segregation ratio can be obtained by counting individuals with and without expression of the target gene, while the population genotypic segregation ratio can be obtained by counting individuals with and without the target gene in the T1 generation of transgenic plants. The former method is often used when estimating the number of transgene insertions. However, the results obtained by phenotyping are often lower than the actual situation, while the results obtained by genotyping can accurately determine the number of transgene insertions. Comparison of the results of the analysis of the T0 generation and its progeny can also provide information on the number of transgene insertion sites. In recent years, the precise assessment of the number of transgene insertions has played an important role in the development of the technique of "gene removal", which consists in the co-transformation of several T-DNA regions to obtain transgenic plants free of any selective marker gene. The number of insertion sites of an exogenous gene in a transgenic plant does not correspond exactly to the number of copies of the exogenous gene, because it is possible to have multiple copies of the same insertion site, or to have a single copy inserted into several different sites.
1. segregation of transgenic phenotypes
The T0 generation of transgenic plants can be selfed or crossed to obtain the T1 generation, and the expression of the exogenous gene can be evaluated using the generation of plants. Any exogenous gene that has been transferred into the plant genome can be analyzed by the phenotype of the offspring, and screening for selective marker genes in T0 transgenic plants is often applied to identify the expression of exogenous genes in T1 transgenic plants. If the phenotype of the target gene can be easily observed, it can also be used to analyze the phenotype of several generations of transgenic plants, e.g., the phenotype of genes related to starch synthesis can be easily observed by iodine staining test. Antibiotics such as kanamycin and thiamphenicol can also be used to identify positive plants containing relevant resistance genes such as the kanamycin and thiamphenicol genes, respectively, in the transgenic progeny (see Note 1). Alternatively, transgenic plants containing functional genes such as BAR, ALS, or EPSP can be rapidly identified by spraying seedlings or coating leaves with herbicides such as glufosinate, sulfonylurea, or glyphosate. In contrast, reporter genes such as β-glucuronidase gene, LUC and green fluorescent protein gene are usually used to detect exogenous gene expression in NaiGen plants. Non-invasive phenotyping, such as observing the expression of the green fluorescent protein gene or LUC, is preferable to invasive phenotyping, such as staining for the glucuronidase gene or resistance assays, because the former can be used for subsequent molecular assays in surrogate plants in which the exogenous gene is not expressed.
In events 1 and 2, where generation transgenic plants were obtained from selfing of T. generation plants, their β-glucuronidase gene activity was determined mainly by histochemical staining of endosperm, leaves, and roots.
( 1 ) For endosperm staining, the seeds were first sterilized with 70% ethanol for 30 s, then soaked in 50% saturated sodium hypochlorite solution for 15 min, then washed with sterile deionized water for three times, and then rinsed for the third time and soaked for several hours. The treated seeds were cut into two halves with a sterilized scalpel on an ultraclean bench, and the embryo half of the seeds was used to obtain Naikai seedlings on MS medium, while the other half was used for β-glucuronidase (GUS) staining. The phenotypes of endosperm and embryo are often compared together because they have the same genotype but different genomic ploidy.
( 2 ) For root and leaf staining, it is not necessary to sterilize the seeds; of course the seeds can be washed in 70% ethanol for 30 s and then three times in sterile deionized water. For some species, such as wheat and barley, the seeds are first incubated on moist filter paper at 5°C for 2-4 days in the dark, followed by germination at 20-25°C for 5 days in the dark, and then incubated at the same temperature with 16 h of light per day, and the roots and leaves are collected after 5-7 days. This method is preferred to endosperm staining because the seeds of some species (e.g. wheat and barley) are very sensitive to sodium hypochlorite.
( 3 ) Depending on the size of the samples, they are submerged in 96-, 24-, or 6-well plates with β-glucuronidase reaction solution.
( 4 ) The samples were placed on open plates under vacuum (71 mm Ley column) for 10-15 min, and the reaction was carried out overnight at 37°C on a shaker.
( 5 ) The next day, observe the β-glucuronidase-stained samples. Fig. 13.1 shows the result of endosperm staining of T1 generation transgenic seeds in Example 2. 
( 6 ) Estimation of transgenic insertion site: A segregation ratio can be derived from the observation of β-glucuronidase activity in T1 transgenic plants. Based on the chi-square analysis between the observed values and the Mendelian theory of inheritance of a single gene (Table 13.1), the number of insertion sites of a single gene can be determined (Table 13.2). In Example 2, 83 out of several generations of transgenic plants had β-glucuronidase (GUS) activity, whereas 17 did not (Figure 13.1), a result that suggests that the transgene insertion site should be a single one. Although this method of analysis has been used in much of the literature, it is worth noting that the results from this method are only an estimate of the transgene insertion site (see Note 2). Also in Example 2, the results of further molecular testing (see "Genotype-based genetic analysis" in Section 3.1) indicate that there should in fact be two separate insertion sites for this subexogenous gene, not one, and that the results based on the phenotype are therefore inaccurate. Therefore, the real genetic identification should be obtained based on the analysis of the results of the investigation of the genotype of the transgenic progeny and not the phenotype. 
2. Genetic analysis based on genotype
The theoretical ratios of segregation according to Mendelian laws of inheritance in segregating populations of progeny after one self-crossed generation when one or two genes are inserted into one, two, three or four loci are presented in Tables 13. 1 and 13. 3, respectively. Nai generation plants were the object of study, and the analytical methods used were molecular assays such as PCR assay or dot hybridization. Since genotyping of several generations of plants by molecular means is more expensive and labor-intensive than phenotyping (see Section 3.1, "Segregation of transgenic phenotypes"), genotype-based genetic analysis is generally rarely used, despite the limitations of phenotype-based analysis (see Note 2). Of course, it is preferable to combine the two approaches when possible. In the following, we will describe the procedure for such an analysis in the context of Example 2: 
( 1 ) The cultivation of transgenic plants and phenotyping of segregation results are performed according to steps 2 to 5 in section 3.1 on segregation of transgenic phenotypes. An important step to ensure the accuracy of the results is that naira plants are not selected so that all plants survive and can be used for subsequent experimental analysis. This is because screening for antibiotics or herbicides can kill transgene-silenced individuals and thus bias the results of the genetic analysis (see Note 2 ). In Example 2, the 83 T1 generation seeds with significant β-glucuronidase activity (Fig. 13. 1) should have contained the β-glucuronidase gene and therefore did not need to be analyzed further. On the other hand, the 17 generations of plants without β-glucuronidase activity should be further characterized by molecular assays to clarify whether they were silenced by the β-glucuronidase gene or did not contain the β-glucuronidase gene and did not show β-glucuronidase activity.
( 2 ) Collect leaf samples from 17 surrogate plants without β-glucuronidase activity, as well as negative control (wild-type plants) and positive control samples detected by PCR, the length of the samples should be matched with 1.5 ml microcentrifuge tubes.
( 3 ) Extract genomic DNA from the leaf samples with the DNA extraction kit according to the instructions of the product.
( 4 ) The amount of DNA template to be added to the PCR reaction depends on the genome size of each species. 25 μl of rice genomic DNA is usually 25 ng in the PCR reaction system, while 100-150 ng for barley or wheat.
( 5 ) Each PCR system includes plant genomic DNA, 1x reaction buffer (containing 2.5 mmol/L MgCl2 ), 250 μmol/L dNTP, 0.4 μmol/L each of forward and reverse primers (e.g., according to the design of β-glucuronidase gene), and 2 U of Taq DNA polymerase.
( 6 ) PCR reaction procedure: the DNA samples were denatured at 95°C for 1 min, followed by denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min, for a total of 30 cycles; at the end of the cycle, the samples were finally extended at 72°C for 10 min.
( 7 ) PCR reaction products (5~10 μl) were analyzed by electrophoresis on 1%~1.2% (m/V) agarose gel containing ethidium bromide (5 μl of ethidium bromide was added to every 100 ml of gel, and the agarose gel was heated and dissolved with 0.5X TBE buffer), and electrophoresis was carried out with a voltage of 80~100 V for about 20 min. 17 samples of the generation test had 7 single samples, which were detected by the UV lamp (Figure 13.2). Under the UV light (Fig. 13.2), it was observed that 7 out of 17 samples containing the glucuronidase gene had no β-glucuronidase activity (i.e., the β-glucuronidase gene was silenced).
( 8 ) DNA samples can be quality characterized by repeating steps 5-7 using primers for the single-copy housekeeping genes or RFLP probes of the plant homeodomain to ensure that they can be successfully amplified by PCR. Analysis of the amplification of the housekeeping genes showed that the 10 samples obtained from Example 2 could be amplified by PCR (Fig. 13. 2).
( 9 ) Calculation of the number of transgene insertion sites: Using the theoretical values of the Mendelian laws of inheritance ( Table 13. 1 ) it was possible to perform a chi-square test on the observations obtained. In Example 2, 90 T1 generation monocots contain β-glucuronidase gene, while 10 monocots do not. Based on the chi-square test results, we can see that the insertion site of the transgene in the genome of the plant should be two, which can be independently and freely segregated (Table 13.2).
3. Cross-generation Southern analysis
Southern analysis is generally used to characterize the arrangement of the insertion sites of individual transgenes (see section 3.2 "Southern analysis"). Of course, it can also be used to determine the number of transgene insertion sites by comparing the results of crosses between transgenic plants of different generations. For example, the number of insertion sites of the exogenous gene can be determined from the results of the Southern analysis of the T-generation and of the plants of the previous generation obtained by self-crossing. This method is described in the following, using Example 2 as an example. 
( 1 ) The DNA of the sample can be extracted from the leaves of the plant according to the method recommended in the DNA Extraction Kit (see Note 3), and the concentration of the DNA is calculated from the absorbance value of the sample at a wavelength of 260 nm. Samples can be stored at 4°C for a short period of time or at -20°C for a long period of time (see Note 4).
( 2 ) The amount of DNA to be used for Southern analysis depends on the size of the genome of each crop, generally 5 μg for rice, and 20 μg for barley and wheat. 50-80 μl of reaction volume is required for DNA digestion, and the amount of restriction endonuclease is 10-15 U/μg of DNA (see Note 5). In addition, the DNA of the negative control (i.e., wild type) and the positive control (e.g., a single strain that has been identified as positive by Southern analysis) should be digested at the same time when performing DNA digestion of the samples.
( 3 ) The digested reaction solution should be concentrated to a volume of about 30 μl by vacuum centrifugation, and then added to an agarose gel at a concentration of 0.8% (melted with 1x TBE) and electrophoresed overnight at 40 V. At the same time, 1x TBE should be added to the gel. One or two markers (e.g., 600 ng of λ-phage DNA product digested with Pst I or Hind III) should also be added to the gel. In addition, restriction enzyme reaction buffer should be added to the blank wells to avoid possible band bias during electrophoresis.
( 4 ) After electrophoresis, the gel stained with ethidium bromide is photographed under a UV light to determine the extent of DNA digestion and the homogeneity of the individual samples, and the position of the labeled bands in the gel is recorded.
( 5 ) The gel was first washed with 0.25 mol/L HCl solution (shaking slowly) for 15 min and then rinsed with water, then denatured in 0.4 mol/L NaOH solution for 15 min, and then blotted the DNA samples onto the nylon membrane overnight. After blotting, the nylon membrane was washed in 2X SSC solution for 2 min, and then wrapped in a film and stored at 5℃ for short-term storage or -20℃ for long-term storage.
( 6 ) The nylon membrane was prehybridized in hybridization solution for 2-5 h at 65°C with slow shaking. 50 ml of hybridization solution contained 2 ml of 5x HSB, 1 ml of Denhardt's reaction solution II, and 1 ml of carrier DNA (denatured in boiling water for 7 min).
( 7 ) The probe for hybridization (approximately 500 bp in length) can be obtained either by gel recovery of the transformed plasmid by enzymatic digestion (QIAquick Gel Extraction kit), or by PCR amplification followed by PCR recovery kit (QIAquickPCR Purification kit). The procedure was performed according to the instructions of the product. 25 ng (15 μl by volume) of probe was boiled in a water bath for 7 min and then placed on ice for 5 min, then 5 μl of OLB, 2 μl of BSA, 2 μl of DNA Polymerase I Klenow Fragment (5 U/μl), and 2 μl of 32P dCTP were added and reacted at 37℃ for 2 h to complete the labeling of the probe. The labeled probe was denatured with 2.6 μl of 3 mol/L NaOH for 5 min and then added to the prehybridization solution. The hybridization membrane was slowly shaken in the prehybridization solution at 65 ℃ overnight.
( 8 ) The nylon membrane was washed twice with 500 ml of 2x SSC and 1% SDS solution for 15 min each time, and then washed twice with 500 ml of 0.2 X SSC and 1% SDS solution for 15 min each time.After washing, the nylon membrane was wrapped with film.
( 9 ) Estimation of the number of insertion sites of the transgene: The processed nylon film can be analyzed by film autoradiography or phosphorimage analysis. For example, in Example 2, two insertion sites were identified by comparing the results of Southern analysis of T0 and T1 generation plants (Fig. 13.3). Although the Southern analysis method can accurately identify the number of transgenic insertion sites, it has its limitations, as described in Note 6. In addition, this analysis can also be used to test the stability of the structure of the transgene locus in the progeny. When multiple DNA fragments or vectors are transformed together, Southern analysis should also be performed with a single probe that can be used in common or with a mixture of multiple specific probes (i.e., one probe per fragment or vector). 
Conformation of each transgene insertion site
When there is only one insertion site of the exogenous gene, the copy number and arrangement of the exogenous gene at the insertion site can be understood through molecular characterization of T. generation plants. When multiple insertion sites are found in transgenic plants (identified according to the methods in Section 3.1), each insertion site should be analyzed one by one, mainly by analyzing the single plants of the segregating population or by analyzing the pure lines of each site.
1. Southern analysis
Southern analysis is the most commonly used method to characterize the configuration of a transgene insertion site. The entire structure of each insertion site can be reconstructed by a combination of enzymatic digestion and probes that cover the full-length DNA sequence used for transformation, including the backbone sequence of the vector. The first step is to estimate the number of copies of the exogenous gene at each insertion site (either as is or rearranged). Depending on the complexity of the insertion site different methods are used for the analysis. The following describes the specific methods for analyzing simple transgenic insertion sites containing one or a few copies, and complex transgenic insertion sites containing a large number of copies, respectively.
( 1 ) DNA extraction, membrane preparation, and hybridization can be described in Section 3.1, Steps 1 to 8. Plant genomic DNA is usually cleaved by a single enzyme, in this case the Xbal cleavage site in the plasmid. The probe used must be able to hybridize specifically to the exogenous gene sequence at the 5' (in the case of the thaumatin gene) or 3' (in the case of the β-glucuronidase gene) end of the cleavage site (see Note 8).
( 2 ) Preliminary analysis of the membrane with a radioautoradiograph or photographic imager will generally provide a preliminary assessment of the complexity of each insertion site based on the results obtained.
① Analysis of simple insertion sites
After digestion and probe hybridization, the results of Southern analysis yield up to 4 hybrid bands of similar brightness (e.g., Example 2, sites 1 and 2).
a. The restriction endonucleases (see Note 9) and probes used for Southern analysis (Steps 1-8 in Section 3.1) can be found in Table 13.4 for common methods. Southern analysis allows not only the study of sequences within the region of the transgenic locus, but also the analysis of sequences in close proximity on both sides. If more precise results are required, multiple analyses with different enzymes and probes are recommended. For example, in locus 1 of Example 2 (Fig. 13.4), the site should be cleaved again with Hind III as a restriction endonuclease, which is located on the side of the expression region of the β-glucuronidase gene, and hybridization should be performed with the sequences of the promoter or the terminator of the β-glucuronidase gene as a probe, since multicopies and rearrangements of exogenous genes may be present at this insertion site. Alternatively, the use of probes that bind specifically to vector resistance gene sequences also allows detection of the presence of vector sequences in transgenic individuals. 
b. Structural characterization of site 1 in Event 2: After Xba I and Hind III digestion and hybridization with the β-glucuronidase gene probe, multiple bands appeared on the membrane indicating that the insertion site may contain multiple copies of the β-glucuronidase gene, one intact and one possible deletion. However, there was only one hybridization band between the thaumatin gene and the NPTI gene probe, a result that indicates that the vector sequence has been transfected and integrated into the plant genome. This is mainly caused by ineffective recognition/ shearing of the left border of the T-DNA, resulting in a "read-through" of the T-DNA sequence (see Note 10). Based on the size of the hybridization bands, the structure formed by the insertion site sequence can then be inferred (Fig. 13.4).
c. Structural characterization of event 2, locus 2: Hybridization with the probes for the β-glucuronidase gene and the thiamphenicol gene results in only one band. There is no signal when hybridizing with the probe of β-glucuronidase gene and thiramycin gene, indicating that the vector backbone is not integrated into the plant genome. Based on the size of the hybridization bands, the structure formed by the sequence of the locus can be deduced (Fig. 13.5).
② Analysis of complex insertion sites
Southern analysis of Example 1 using a variety of restriction endonucleases and probes revealed four or more bands with different signal intensities (Figure 13.6). In this case, the hybridization results are generally not very reliable [ 17 ], and the results alone cannot be used to accurately infer the conformation of the insertion site and the copy number of the exogenous gene (see Note 11). However, it is possible to estimate the copy number of the transgene as well as the complete expression unit of the exogenous gene (i.e., promoter+gene+terminator) by analyzing the optical density of the hybridization signal. The results obtained from Southern analysis of an enzyme cleavage site that is not present in the transgene vector can also help to understand the presence of the plant genomic sequence at the insertion site. 
( a ) DNA extraction can be performed as described in Section 3.1, Step 1. The genomic DNA concentration of the wild-type plant and the plasmid DNA concentration of the marker must be determined accurately to facilitate subsequent optical density analysis (see Note 12).
( b ) DNA digestion can be referred to in 3.1 Step 2. A standard sample equivalent to 1, 2, 5, 10, 20, 40 and 80 copies of the exogenous gene is added to 5 μg of wild-type DNA (see Note 13), and it is also recommended to add a positive control to each membrane (see Note 14).
( c ) Southern analysis is performed in steps 3 to 8 of section 3.1, using the Hind III cleavage site adjacent to the β-glucuronidase gene expression region or enzymes without cleavage sites on transformation vectors such as Pac I, Nhe I, Bst X I, Apa I, and Kpn I to degrade the genomic DNA of the plant, and four different probes are required for the analysis: a single-copy RFLP probe; three separate RFLP probes; and three different probes for each of the three genomic DNAs. Four different probes are required for the analysis: a single-copy RFLP probe and three probes that bind specifically to the β-glucuronidase gene, the thaumatin gene, and the NPTI gene, respectively, and the RFLP probe is used to ensure homogeneity in the amount of samples spiked from one sample to the next (see Note 14). After hybridization by Hind III cleavage, some bands are similar in size to the expression region (2.9 kb) of the plasmid DNA cleavage (CfAS), while other larger or smaller bands may be due to DNA rearrangements. Southern analysis using Spe I and HPT gene probes yielded similar results as above (Figure 13.6). The β-glucuronidase gene probe hybridized to two bands when digested with Apa l, a vector with no cleavage site, suggesting that part of the plant genome may also be contained within this insertion site. The presence of a large number of copies of the NPT I gene in Case 1 also confirms the integrity of the vector sequence (since the gene gun method uses an intact vector plasmid). However, based on this information, it is not possible to derive a conformation for the transgene insertion site in Example 1. 
( d ) Identification of transgene copy number in Case 1: The intensity of all hybridization signals on the hybridization membrane after removing the background can be quantitatively analyzed by instruments such as optical densitometer (Bio-Rad 690) or photographic imager. Based on the correlation between signal intensity and gene copy number, a standard curve can be reconstructed by regression analysis. The number of copies of the exogenous genes in each sample can be estimated from the results of the standard curve and the enzyme-probe combination (see Note 15). The signals from the Hind III, Spe l, and Xba I enzyme crosses indicate that transgenic Example 1 contains 36 copies of the β-glucuronidase gene and 14 copies of the thaumatin gene (Fig. 13.6).
2. Other methods
Through Southern hybridization analysis, a general understanding of the overall structure of each transgene insertion site was obtained. However, to fully grasp all the information of the transgene insertion sites, sequencing analysis of the insertion region and the genomic sequences of the immediate neighbors is required [18, 19 ], and sometimes the sequence analysis of the genomic DNA is more important. In this chapter, this method will not be described in detail; however, high-throughput analysis methods of transgenic near-neighbor sequences of model plants such as Arabidopsis thaliana [ 20 , 21 ] and rice [ 22 ] have been reported. Based on the sequencing results of the genomic sequences of plants in the immediate vicinity of the insertion region, PCR primers can be designed to amplify and sequence the entire insertion region step by step. In addition, primers designed according to the proximate sequences can be used for amplification of wild-type genomic sequences, which can identify whether there is a loss of genomic sequence at the insertion site of the exogenous gene. At the same time, DNA sequencing can also identify whether co-integration of chloroplast DNA and exogenous genes occurs during transgenesis.