A method for non-invasive rapid screening of chicken go generation chimera
By integrating the Gluc gene into chicken PGCs and detecting Gluc activity in blood samples, the problem of the inability to quickly and non-invasively detect chicken G0 generation chimeras in existing technologies has been solved, enabling early and efficient screening and sex-specific assessment, and reducing breeding costs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- XIANGHU LABORATORY
- Filing Date
- 2026-04-20
- Publication Date
- 2026-06-12
AI Technical Summary
Existing technologies cannot quickly and non-invasively detect the degree of chimerism of exogenous PGCs in chicken G0 generation chimeras, resulting in low screening efficiency, high cost, and inability to assess chimerism status in the early stages.
The secretory luciferase gene was integrated into the genome of chicken PGCs, and G0 generation chimeric chicks were prepared by intravenous injection into the embryo. The activity of luciferase in trace blood samples was detected to assess the degree of chimerism of exogenous PGCs.
It enables early, non-invasive, and highly sensitive chimera screening, shortens the screening cycle, reduces costs, is suitable for sex-specific assessment in roosters, and is easy to operate.
Smart Images
Figure CN122189153A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a non-invasive rapid screening method for chicken G0 generation chimeras, belonging to the field of biotechnology. Background Technology
[0002] Currently, the construction of genetically modified animals mainly relies on techniques such as somatic cell nuclear transfer, pronuclear microinjection of fertilized eggs, embryonic stem cell chimerism, and primordial germ cell (PGC) chimerism. In establishing genetically modified models in poultry such as chickens, PGC chimerism is the primary strategy. However, unlike rodent chimeras prepared using embryonic stem cells (whose chimerism can be visually identified through surface features such as feather color), G0 generation chimeric chickens obtained through PGC chimerism have exogenous PGCs distributed only in the gonadal tissue. This tissue-specific chimerism pattern makes it impossible to directly assess the degree of chimerism through external phenotypes, thus creating a significant technical bottleneck for early identification. Current mainstream detection methods have obvious limitations: firstly, they require dissection of G0 generation individuals to obtain gonadal tissue for molecular biological testing; secondly, they require waiting for G0 generation individuals to reach sexual maturity (approximately 6 months) to collect gametes (such as semen) for subsequent analysis. These methods are either invasive and destructive or time-consuming. During the period of waiting for sexual maturity, a large number of G0 generation chicks need to be raised to screen for effective chimeric individuals, which significantly increases the feeding cost and causes a waste of resources. Therefore, developing a method that can quickly and non-invasively detect the chimerism of exogenous PGCs in G0 generation chimeric chickens at an early stage is of great and urgent significance for improving the efficiency of avian gene editing breeding and reducing research and development costs. Summary of the Invention
[0003] The purpose of this invention is to address the shortcomings of existing technologies by proposing a non-invasive, rapid screening method for chicken G0 generation chimeras. This enables early and rapid screening of chicks with a high chimerism rate, facilitating rapid breeding.
[0004] Gluc (Gaussia princeps luciferase) is a highly sensitive and stable bioluminescent enzyme derived from the marine copepod Gaussia princeps. It catalyzes the emission of bright blue light from the conjugated enzyme substrate coelenterin. Gluc has a small molecular weight of approximately 20 kDa and contains a signal peptide sequence, allowing it to be efficiently secreted from mammalian cells into culture media or blood (Tannous et al., 2005; Wurdinger et al., 2008). The Gluc reporter system has been used in studies to detect protein secretion rates (Hewett et al., 2007; Suzuki et al., 2007; Badr et al., 2007), promoter activity (Ruecker et al., 2008; Nishimura et al., 2013), and in vivo cell proliferation (Sugawara et al., 2023). Furthermore, the half-life of the Gluc protein in blood is only 20 minutes (Tannous, 2009), making this system suitable for real-time observation of cells in vivo. Based on the above research, this invention integrates the secretory luciferase Gluc gene into the genome of chicken PGCs, prepares G0 generation chimeric chicks through intravenous injection into the embryo, and detects Gluc activity in trace blood samples. This allows for non-invasive assessment of the chimerism of exogenous PGCs without damaging cells or tissues, enabling convenient and rapid screening of chicks with high chimerism rates after birth for subsequent feeding and breeding, thus accelerating the breeding process of genetically modified chickens.
[0005] The present invention solves the technical problem through the following technical solution: First, a non-invasive and rapid method for screening chicken G0 generation chimeras is provided, including the following steps: Step 1: Provide chicken primordial germline stem cells expressing secretory luciferase; Step 2: Inject the chicken primordial germline stem cells from Step 1 into the recipient embryo, and hatch to obtain G0 generation chimeric chickens; Step 3: Detect the activity of secretory luciferase in the peripheral blood of the G0 generation chimeric chickens from Step 2; Step 4: Assess the degree of chimerism of in vivo and exogenous primitive reproductive stem cells in G0 generation chimeric chickens based on the results of secretory luciferase activity detected in Step 3.
[0006] In step one of the above method, the secretory luciferase is Gaussian princeps luciferase (Gluc). The chicken primordial germ cells expressing the secretory luciferase in step one are obtained by introducing an expression vector containing the secretory luciferase gene into the primordial germ cells; the expression vector is a piggyBac transposon vector; the piggyBac transposon vector further contains a promoter operatively linked to the secretory luciferase gene, the promoter being the EF1a promoter. The piggyBac transposon vector also contains a reporter gene, the reporter gene being the GFP gene, linked to the secretory luciferase gene via an IRES sequence.
[0007] Furthermore, in step two, the recipient embryo is a chicken embryo that has developed to 48-56 hours.
[0008] Furthermore, in step three, peripheral blood samples are collected between 20 and 60 days after chicken hatching; the method for detecting secretory luciferase activity is to contact the peripheral blood sample with coelenterate substrate and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the bioluminescence value.
[0009] Furthermore, the evaluation in step four includes screening for highly chimeristic individuals by comparing the levels of secretory luciferase activity among different G0 generation individuals.
[0010] Furthermore, the chicken G0 generation chimera is a rooster.
[0011] Secondly, a chicken primordial germline stem cell expressing secretory luciferase is provided, wherein the stem cell is obtained by introducing an expression vector containing the secretory luciferase gene into the primordial germline stem cell.
[0012] Finally, the application of chicken primordial germline stem cells expressing secretory luciferase in the preparation of genetically modified chickens is presented.
[0013] The beneficial effects of this invention are as follows: First, this method achieves non-invasive detection, requiring only a small amount of peripheral blood, eliminating the need for dissection or waiting for individual sexual maturity to collect gametes, thus causing no trauma to the experimental animals. Second, it has the capability for early and rapid screening, allowing detection in early stages after chick hatching (e.g., 55 days of age), significantly shortening the screening cycle and effectively avoiding the high costs associated with raising large numbers of ineffective individuals. Third, it exhibits high sensitivity and stability: employing the Gluc reporter gene system, it boasts high sensitivity, and the piggyBac transposon system-mediated transgene expression in vivo demonstrates continuous and stable characteristics, ensuring the reliability of the detection results. Fourth, it enables sex-specific assessment: this method shows significant detection efficiency in roosters, consistent with the biological characteristic of low colonization efficiency of male primordial germ cells (PGCs) in female recipients, providing an important basis for screening effective chimeras. Finally, the operation is simple, requiring no complex instruments or equipment, and is easy to promote in practical applications. Attached Figure Description
[0014] Figure 1 The successful establishment of Hy-Line White chicken PGCs. Part A of the figure shows the morphology of Hy-Line White chicken PGCs cultured in the mKO-F system, with a scale bar of 400 μm; Part B of the figure shows the expression of the SSEA-1 protein, an antigen on the membrane surface of PGCs, detected by immunofluorescence staining, with a scale bar of 200 μm.
[0015] Figure 2 Stable PGCs integrating Gluc and GFP were successfully constructed. Part A in the figure shows the EF1a promoter initiating the Gluc-IRES-GFP element. piggyBac The transposon plasmid vector and the PB transposase expression vector for CAG-initiated expression are shown in Part B. Part B shows the morphological images of PGCs after flow cytometry sorting. The left image shows the morphological images of PGCs under bright field conditions, and the right image shows PGCs labeled with green fluorescent dye. The scale bar is 200 μm. Part C shows the content of Gluc in the supernatant of PGCs-Gluc IRES GFP cells cultured after flow cytometry sorting and the supernatant of ordinary PGCs cells without transfection plasmid.
[0016] Figure 3 The results of observing GFP fluorescence in gonadal tissue under a fluorescence microscope on the 12th day after birth are shown. The left set of images shows gonadal tissue with wild-type unintegrated GFP, and the right set of images shows gonadal tissue with PGCs containing stably integrated Gluc-IRES-GFP elements.
[0017] Figure 4The results of detecting Gluc protein activity in the blood of G0 generation chicks. Part A of the figure shows a representative image of G0 generation roosters expressing GFP and Gluc in their gonads; Part B shows the measured Gluc content in the blood of various chimeric chicks 55 days after birth; Part C shows the measured Gluc content in the blood of G0 generation chimeric chicks on day 120 after birth. Detailed Implementation
[0018] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.
[0019] Example 1 Preparation of chicken primordial germline stem cells expressing Glu PGCs isolation and cultivation From 20 Hainan White chicken hatching eggs that had been incubated for 7 days, the mKO-F system (mKO medium was prepared based on the mKO medium developed by Lu Yangqing's research group, the components of which included self-made avian KO-DMEM (AKO) medium, 0.2% chicken serum (Aladdin, C774393), 1% fetal bovine serum (Gibco, 10099), 1.2 mM sodium pyruvate (Thermfisher, 11360070), 100 ug / ml heparin sodium (Aladdin, H104201), 1X antibiotic-antimycotic (Thermfisher, 152470-062), 1X GlutaMax (Thermfisher, 35050), 1X GS nucleosides supplement (EmbryoMax, ES-008-D), 0.1 mM β-Mercaptoethanol (SIGMA, M3148-25ML), 4 ng / ml hFGFb (R&D, 234-FSE-025), 1X NEAA (Gibigo, 11140-050), 1X B-27 (Thermofisher, 17504044), 25 ng / ml Human Activin A (Peprotech, 120-14-10). The self-made 50 mL AKO medium consisted of 11.6 mL ultrapure water for cell culture, 37.5 mL calcium-free DMEM medium (Seville, G4519), 0.5 mL 50 mM MEM amino acid solution (Thermfisher, 11130051), 0.5 mL 100 mM sodium pyruvate (Thermfisher, 11360070), and 0.5 mL... 100 μM MEM vitamin solution (Thermfisher, 11120052). F represents feeder cells, obtained from BRL cells after treatment with mitomycin C. Primitive germ cells were isolated from gonadal tissue. Eighteen chicken primordial germ cell lines (PGCs) were successfully isolated, with a success rate of approximately 90%. These cells exhibit typical morphological characteristics of chicken PGCs: suspension growth, relatively large size, most cells are round or oval, and the cytoplasm contains glycogen granules and lipid droplets. Figure 1 Part A). Immunofluorescence staining targeting SSEA-1 protein revealed that these cells highly expressed SSEA-1 (part A). Figure 1 (Part B) confirmed that these cells are pluripotent PGCs.
[0020] Construction and transfection of expression vectors Construct a piggyBac transposon vector containing a Gluc-IRES-GFP element expressed by the EF1a promoter. Figure 2Part A of the vector was co-transfected with the transposase expression vector into cultured male PGCs using conventional electroporation. After approximately one week of culture, once the exogenous gene had been stably integrated, GFP-positive PGCs were sorted using flow cytometry and further enriched to a sufficient quantity. Figure 2 Part B).
[0021] Gluc expression and secretory activity verification Supernatants from wild-type PGCs and transfected PGCs-Gluc IRES GFP cells were collected. After adding coelentrin substrate, the luminescence values were measured using a microplate reader. The results showed a strong luminescent signal in the transfected cell supernatant, indicating the presence of a large amount of active Gluc protein that could be secreted extracellularly; however, no luminescent signal was detected in the wild-type PGCs cell supernatant. Figure 2 (Part C of the protein). This indicates that the Gluc protein in the constructed cells can be expressed and secreted normally.
[0022] Example 2 Preparation of G0 generation chimeric chickens The GFP-positive PGCs enriched and cultured in Example 1 were injected into recipient embryos that had been developing for 52-54 hours using microinjection combined with the open-window method. The injection details and hatching results are shown in Table 1.
[0023] Table 1. Statistics on the injection and hatching of PGCs simultaneously labeled with GFP fluorescence and Gluc.
[0024] The results showed that the hatching rate was approximately 22% after injection using the open-window method. On the 12th day after hatching, gonadal tissue was extracted from some chicks and observed under a fluorescence microscope. The results showed that the chimeric rooster's testes contained a large number of green fluorescent cells derived from exogenously injected PGCs, while no GFP-positive cells were observed in the wild-type gonadal tissue. Figure 3 This indicates that the exogenously injected PGCs successfully chimeric into the gonads of the recipient chicken.
[0025] Example 3 Peripheral blood Gluc activity detection and chimerism assessment All 18 hatched G0 generation chimeric chickens ( Figure 4 Part A) was used for live sampling analysis.
[0026] Early detection (day 55) Peripheral blood samples were collected from chicks on day 55 post-hatching. After processing the blood samples with coelenterate substrate, luciferase activity was quantitatively detected using a microplate reader. Results showed ( Figure 4(Part B): High levels of Gluc activity were detected in blood samples from all rooster chimeras, with significant inter-individual differences; in contrast, Gluc activity in blood samples from hen chimeras was close to background levels. This significant sex difference is consistent with the biological characteristics of male-derived PGCs having lower migration and colonization efficiency in the female recipient gonads.
[0027] Expression stability test (day 120) To assess the in vivo expression stability of PB transposon-mediated Gluc transgenes, blood samples were collected again from all surviving chicks approximately 120 days post-hatching, and Gluc activity was measured. Results showed that the overall pattern of Gluc activity at 120 days post-hatching was consistent with that at 55 days post-hatching. Figure 4 (Part C of the spectrum). Furthermore, the Gluc activity at 120 days post-incubation was approximately five times that detected at 55 days post-incubation. Figure 4 (Parts B and C). This increase may reflect a corresponding increase in the number of endogenous Gluc transgene-positive germ cells in the recipient gonads, possibly due to the resumption of cell division in spermatogonia around 10 weeks post-hatching. These results indicate that the PB transposon system-mediated Gluc transgene expression is continuous and stable in chickens.
[0028] The above results indicate that a simple, non-invasive peripheral blood glucose activity assay can be used to effectively assess and quantitatively compare the degree of chimerism of exogenous PGCs in G0 generation chicks in vivo, thereby screening out individuals with high chimerism ratios for subsequent feeding and breeding.
[0029] In addition to the above-described embodiments, the present invention may have other implementations. All technical solutions formed by equivalent substitution or equivalent transformation fall within the protection scope claimed by the present invention.
Claims
1. A non-invasive rapid screening method for chicken G0 generation chimeras, characterized in that, Includes the following steps: Step 1: Provide chicken primordial germline stem cells expressing secretory luciferase; Step 2: Inject the chicken primordial germline stem cells from Step 1 into the recipient embryo, and hatch to obtain G0 generation chimeric chickens; Step 3: Detect the activity of secretory luciferase in the peripheral blood of the G0 generation chimeric chickens from Step 2; Step 4: Assess the degree of chimerism of in vivo and exogenous primitive reproductive stem cells in G0 generation chimeric chickens based on the results of secretory luciferase activity detected in Step 3.
2. The method for non-invasive rapid screening of chicken G0 generation chimeras according to claim 1, characterized in that: In step one, the secretory luciferase is Gaussia princeps luciferase Gluc.
3. The method for non-invasive rapid screening of chicken G0 generation chimeras according to claim 1 or 2, characterized in that: In step one, the chicken primordial germ cells expressing secretory luciferase are obtained by introducing an expression vector containing the secretory luciferase gene into the primordial germ cells; the expression vector is a piggyBac transposon vector; the piggyBac transposon vector also contains a promoter operatively linked to the secretory luciferase gene, the promoter being the EF1a promoter.
4. The method for non-invasive rapid screening of chicken G0 generation chimeras according to claim 3, characterized in that: The piggyBac transposon vector also contains a reporter gene, which is a GFP gene, linked to a secretory luciferase gene via an IRES sequence.
5. The method for non-invasive rapid screening of chicken G0 generation chimeras according to claim 1, characterized in that: In step two, the recipient embryo is a chicken embryo that has developed to 48-56 hours.
6. The method for non-invasive rapid screening of chicken G0 generation chimeras according to claim 1, characterized in that: In step three, peripheral blood samples are collected between 20 and 60 days after hatching. The method for detecting secretory luciferase activity is to contact the peripheral blood sample with coelenterate substrate and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the bioluminescence value.
7. The method for non-invasive rapid screening of chicken G0 generation chimeras according to claim 1, characterized in that: The evaluation in step four includes screening for highly chimeristic individuals by comparing the levels of secretory luciferase activity among different G0 generation individuals.
8. The method for non-invasive rapid screening of chicken G0 generation chimeras according to claim 1, characterized in that: The chicken G0 generation chimera is a rooster.
9. A chicken primordial germline stem cell expressing secretory luciferase, characterized in that: The stem cells were obtained by introducing an expression vector containing a secretory luciferase gene into primordial germ cells.
10. The application of chicken primordial germline stem cells expressing secretory luciferase as described in claim 9 in the preparation of genetically modified chickens.