A gene mutation screening method for activating adaptive responses in fruit flies using low-dose pre-irradiation

Abstract

This invention discloses a gene mutation screening method that utilizes low-dose pre-irradiation to activate the adaptive response in fruit flies. This method combines the high precision and controllability of X-ray flaw detectors with the biological principles of the fruit fly's "adaptive response" to construct a sequential procedure of "low-dose pre-irradiation + specific interval recovery + high-dose challenge irradiation." This procedure aims to transform the adaptive response, which typically has a protective function, into a "pre-damage repair - challenge overload" model. This allows the pre-activated or error-prone cellular repair system to introduce a higher frequency of repair errors (i.e., gene mutations) when dealing with large-scale DNA damage, while simultaneously mitigating overall biological damage, ultimately achieving optimization by increasing both mutation rate and survival rate.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a gene mutation screening method that utilizes low-dose pre-irradiation to activate the adaptive response of fruit flies. Background Technology

[0002] Drosophila, as a classic model organism, is often used to study the effects of environmental stress on physiology, behavior, lifespan, and genetics. Radiation is a commonly used stressor; X-rays directly cause DNA strand breaks or base damage through ionization, thereby inducing gene mutations (Hermann J. Muller). Traditional X-ray mutagenesis methods typically use single high-dose irradiation. While inducing mutations, this method also causes widespread and severe cellular damage and even death in the organism, masking some valuable genetic phenotypes. This results in high "background noise" in mutation screening, leading to low survival rates and screening efficiency of effective mutants. Furthermore, the occurrence of mutations is random and unpredictable, making it difficult to achieve efficient and targeted mutation enrichment.

[0003] Radiobiological studies have revealed an endogenous protective mechanism in organisms against radiation damage called the "adaptive response" (Schäppi-Büchi C. On the genetic background of the adaptive response to X-rays in Drosophila melanogaster. Int J Radiat Biol. 1994 Apr;65(4):427-35.). This means that when an organism is given a very low dose of radiation beforehand (conditional dose) and then receives a higher dose (challenge dose) over a period of time, its mortality rate is significantly lower than that of individuals directly receiving the same dose of radiation. Currently, no existing technology combines this biological phenomenon of the "adaptive response" with high-precision irradiation equipment as a controllable tool to enhance the output of specific genetic experiments. Therefore, developing a systematic approach that integrates high-precision irradiation equipment with the biological characteristics of Drosophila, actively regulating its adaptive response to achieve proactive and efficient induction of gene mutations and screening of mutants, and ultimately forming a dedicated method and system for highly efficient mutant screening, has significant innovative value and practical application significance. Summary of the Invention

[0004] Existing X-ray mutagenesis methods suffer from single-irradiation, random and unpredictable mutation occurrence, severe damage, low survival rate and screening efficiency of effective mutants, and uncontrollable operation. This invention provides a highly efficient gene mutation screening method. By combining the high precision and controllability of X-ray flaw detectors with the biological principle of "adaptive response" in Drosophila, a sequential procedure of "low-dose pre-irradiation + specific interval recovery + high-dose challenge irradiation" is constructed. This procedure aims to transform the adaptive response, which typically has a protective function, into a "pre-damage repair - challenge overload" model. This allows the pre-activated or error-prone cellular repair system to introduce a higher frequency of repair errors (i.e., gene mutations) when responding to large-scale DNA damage, while simultaneously mitigating overall biological damage, ultimately optimizing both mutation rate and survival rate.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A gene mutation screening method that utilizes low-dose pre-irradiation to activate the adaptive response in Drosophila includes the following steps: Step 1: Select wild-type fruit flies and collect healthy individuals 8-24 hours after emergence; Step 2: Place healthy male and / or female fruit flies that have emerged 8-24 hours prior to molting into an irradiator for low-dose pre-irradiation. Step 3: After pre-irradiation, the fruit flies are cultured under standard culture conditions for 6 to 24 hours. Step 4: Place the recovered fruit flies back into the irradiator for high-dose challenge irradiation; Step 5: Mating the challenged female flies with untreated male flies, or mating the challenged male flies with untreated virgin flies carrying specific balanced chromosomes or markers, or mating the challenged male flies with female flies, collecting their offspring, and screening for mutants on a large scale in the population.

[0006] Furthermore, in step 1, wild-type fruit flies or fruit fly strains with specific sensitivity to X-ray-induced adaptive responses are selected as starting materials. Wild-type is preferred. Canton S For fruit flies, strains with known, weak heterozygous defects in DNA damage repair pathways (such as non-homologous end joining and homologous recombination repair) can also be selected to expand the mutation spectrum.

[0007] Preferably, for individual fruit flies, healthy individuals at a specific developmental stage are collected, preferably young adults 8-24 hours after emergence, whose reproductive cells are in an active state.

[0008] Furthermore, the low dose described in step 2 is 0.2 mGy to 5 mGy. This dose range is set based on the minimum effective dose (0.2 mGy) for inducing an adaptive response, and is intended to activate the DNA damage warning and repair preparation mechanisms of Drosophila germ cells or somatic cells without causing significant direct genetic damage.

[0009] In one specific embodiment of the present invention, a ChiRad 160C X-ray irradiator is used. During pre-irradiation, the parameters of the irradiator are a tube voltage of 40-80 kV and a tube current of 1-2 mA, with an exposure time of 5 seconds.

[0010] Furthermore, in step 3, the standard culture conditions are a temperature of 24-26℃ and a humidity of 50-60%.

[0011] Furthermore, the high dose described in step 4 is 10 Gy to 50 Gy. This dose is higher than the commonly used dose for conventional single irradiation and is designed to cause significant DNA damage. However, due to the adaptive response activated in step 2, the cell's repair behavior may become "aggressive" or "error-prone" in response to this "challenge," thereby introducing more mutations during the repair process rather than simply protecting the genome.

[0012] In one specific embodiment of the present invention, a ChiRad 160C X-ray irradiator is used, and the parameters of the irradiator during challenge irradiation are tube voltage of 120-160 kV and tube current of 3-5 mA.

[0013] Furthermore, the rearing conditions for mating in step 5 are a temperature of 24-26℃ and a humidity of 50-60%.

[0014] In this invention, the mutants in step 5 can be changes in eye color, wing shape, or bristle arrangement, or recessive lethal mutations or loss-of-function mutations in specific pathways can be screened using molecular biology methods (such as deep sequencing of specific loci).

[0015] The method of this invention establishes a reproducible and quantifiable standard operating procedure, facilitating its application in different laboratories. This invention, through a "pre-activated" cell repair system, directs subsequent high-dose irradiation-induced DNA damage more towards the "error-prone repair" pathway, thereby generating a higher local mutation density in the target genomic region (especially in actively repaired regions) than with traditional single high-dose irradiation.

[0016] Low-dose pre-irradiation itself is almost non-toxic, while the activated adaptive response can buffer the acute cell damage caused by high-dose challenge irradiation to a certain extent, improve the survival rate of mutant individuals, and increase the size of the selectable population.

[0017] This invention may preferentially induce mutations in gene networks involved in DNA damage responses, providing a more enriched library of target mutations for studies on genome stability, cancer biology (Drosophila tumor models), and screening of related drugs (anti-radiation or radiation sensitizers). The resulting mutant library can be used in multiple fields such as functional genomics, genetic toxicology assessment, aging research, and the construction of Drosophila models simulating human diseases. Attached Figure Description

[0018] Figure 1 This is a flowchart of the method of the present invention.

[0019] Figure 2 The results are the screening results of the F1 generation mutants in Example 1.

[0020] Figure 3 The results show the survival rate of the F1 generation in Example 1.

[0021] Figure 4 The results show the screening results of the F2 generation mutants in Example 2. Detailed Implementation

[0022] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0024] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Example 1

[0025] Utilizing wild type Canton S Screening for new eye color mutations in fruit fly strains Materials: Wild type Canton S The strain is *Drosophila melanogaster*, and the female flies are observed 24 hours after emergence. The instrument used is a ChiRad 160C X-ray irradiator (Dandong Aolong X-ray Instrument Group).

[0026] Pre-irradiation: 1000 female flies were placed in a breathable sample box and positioned at the center of the irradiator's sample tray. Parameters were set as follows: voltage 60 kV, current 1.5 mA, exposure time 15 seconds. The absorbed dose was measured to be 1 mGy after calibration in the ionization chamber. Irradiation was then performed.

[0027] Recovery: After irradiation, the fly was transferred to fresh culture medium and recovered in an incubator at 25°C for 18 hours.

[0028] Challenge Irradiation: The recovered fruit flies were placed back into the irradiator. Parameter settings: voltage 150 kV, current 4 mA, exposure time 2 minutes, and the measured absorbed dose was 30 Gy. Irradiation was performed using a motorized rotating sample tray to ensure that all samples received a uniform mutagenic dose.

[0029] Genetic design: Treated female flies (F0 generation) were compared with untreated wild-type flies. Canton S Male flies of the strain were mated in batches at a ratio of 3:1.

[0030] Screening: F1 generation males (i.e., male flies that inherited the treated X chromosome from the mother) were collected, and their eye colors (red, orange, light red, white, etc.) were carefully observed under a dissecting microscope. One stable mutant with vermilion eyes was found. Preliminary genetic mapping and sequencing revealed a novel gene related to eye pigment transport. CG10725 ( Figure 2 ).

[0031] Control group: Another group of 1000 Canton S Female flies that received only one 30 Gy irradiation (without pre-irradiation) had a significantly lower F1 generation survival rate than the sequential irradiation group. Figure 3 More importantly, the mutation rate of individuals with stable new eye colors selected in the experimental group was 0.3%, while that in the control group was 0.1%. This indicates that the method of the present invention effectively increases the mutation rate while significantly improving the survival rate of parents. Example 2

[0032] Screening for mutants related to DNA damage repair pathways to construct radiation hypersensitivity models Materials: Drosophila heterozygous strain mus309ᴰ 5 / +。 Mixed male and female flies 24 hours after emergence.

[0033] Irradiation procedure: A sequential procedure similar to that in Example 1 was adopted, with a pre-irradiation dose of 0.5 mGy, a recovery period of 12 hours, and a challenge dose of 15 Gy.

[0034] Screening: The treated fruit flies were crossbred, and individuals exhibiting high lethality to low-dose X-rays (2 Gy) were selected from their F2 offspring. A strain hypersensitive to ionizing radiation was successfully isolated. Whole-genome sequencing identified it as being identical to the maternal strain mus309ᴰ. 5 In addition, it carries a DNA repair gene induced by the method of this invention, located in another DNA repair gene. okra New point mutations ( Figure 4 ).

[0035] Application: This Drosophila strain with dual repair defects can serve as a highly efficient in vivo model for screening radiation sensitizing drugs that can enhance the effects of radiotherapy.

Claims

1. A gene mutation screening method for activating adaptive responses in fruit flies using low-dose pre-irradiation, characterized in that, Includes the following steps: Step 1: Select wild-type fruit flies and collect healthy individuals 8-24 hours after emergence; Step 2: Place healthy male and / or female fruit flies that have emerged 8-24 hours prior to molting into an irradiator for low-dose pre-irradiation. Step 3: After pre-irradiation, the fruit flies are cultured under standard culture conditions for 6 to 24 hours. Step 4: Place the recovered fruit flies back into the irradiator for high-dose challenge irradiation; Step 5: Mating the challenged female flies with untreated male flies, or mating the challenged male flies with untreated virgin flies, or mating the challenged male flies with female flies, collecting their offspring, and screening for mutants on a large scale in the population.

2. The method according to claim 1, characterized in that, In step 1, wild-type fruit flies or fruit fly strains with specific sensitivity to X-ray-induced adaptive responses are selected as starting materials.

3. The method according to claim 1, characterized in that, The low dose mentioned in step 2 is 0.2 mGy to 5 mGy.

4. The method according to claim 1, characterized in that, In step 3, the standard culture conditions are a temperature of 24-26℃ and a humidity of 50-60%.

5. The method according to claim 1, characterized in that, The high dose mentioned in step 4 is 10 Gy to 50 Gy.

6. The method according to claim 1, characterized in that, The breeding conditions for mating in step 5 are a temperature of 24-26℃ and a humidity of 50-60%.

7. The method according to claim 1, characterized in that, The mutants in step 5 include changes in eye color, wing shape, bristle arrangement, and the screening of recessive lethal mutations or loss-of-function mutations in specific pathways using molecular biology methods.

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