The concept of genetic engineering

Genetic engineering refers to strict design according to people's wishes, endowing organisms with new genetic characteristics through in vitro DNA recombination and transgenic technology, and creating new biological types and biological products that are more in line with people's needs. Genetic engineering is designed and constructed at the level of DNA molecule, also known as DNA recombination technology.

(A) the basic tools of genetic engineering

1. "molecular scalpel"-restriction endonucleases (restriction endonuclease)

(1) source: mainly isolated and purified from prokaryotes.

(2) Function: It can recognize the specific nucleotide sequence of double-stranded DNA molecules and break the phosphodiester bond between two nucleotides in a specific part of each chain, so it is specific.

(3) Results: The ends of DNA fragments produced by restriction endonuclease cleavage usually have two forms: sticky end and flat end.

2. "Molecular suture needle"-DNA ligase

(1) Two DNA ligases (e? Large intestine DNA ligase and T4-DNA ligase);

① Similarity: all of them are phosphodiester bond suture.

2 difference: e? Escherichia coli DNA ligase is derived from T4 bacteriophage, which can only connect phosphodiester bonds between complementary sticky ends of double-stranded DNA fragments. T4DNA ligase can suture two kinds of ends, but the efficiency of connecting blunt ends is low.

(2) Similarities and differences between DNA polymerase and DNA polymerase:? DNA polymerase can only add one nucleotide at the end of the existing nucleotide fragment to form phosphodiester bond. DNA ligase connects the ends of two DNA fragments to form phosphodiester bond.

3. "Molecular means of transport"-carrier

The vector (1) has the following conditions: ① It can be replicated in recipient cells and stored stably.

② It has one or more restriction enzyme cut points for inserting foreign DNA fragments.

③ There are marker genes for recognizing and selecting recombinant DNA.

(2) What is the most commonly used carrier? Plasmid is a kind of naked, simple structure, independent of bacterial chromosomes and self-replicating double-stranded circular DNA molecule.

(3) Other vectors: phage derivatives, animal and plant viruses.

(2) Basic operating procedures of genetic engineering

Step 1: Obtain the target gene.

1. The target gene refers to the structural gene encoding protein.

2. Prokaryotic genes are directly isolated, while eukaryotic genes are artificially synthesized. The commonly used methods to synthesize the target gene are reverse transcription and chemical synthesis.

3. PCR amplification of target gene

Principle (1): DNA double-stranded replication

(2) Process: step 1: heating to 90-95℃ to melt DNA; Step 2: cooling to 55-60℃, and binding the primer to the complementary DNA strand; Step 3: heating to 70 ~ 75℃, and synthesizing complementary strand from primer with thermostable DNA polymerase.

Step 2: Construction of gene expression vector.

1. Objective: To make the target gene stably exist in the recipient cells and pass it on to the next generation, so that the target gene can be expressed and play its role.

2. Composition: target gene+promoter+terminator+marker gene.

Promoter (1) is a DNA fragment with special structure, which is located at the head of the gene, where RNA polymerase recognizes and binds, and can drive the gene to transcribe mRNA, and finally obtain the required protein.

(2) Terminator: A DNA fragment with a special structure, located at the end of the gene.

(3) The function of marker gene is to identify whether the recipient cells contain the target gene, so as to screen out the cells containing the target gene. The commonly used marker gene is antibiotic gene.

Step 3: Introduce the target gene into the recipient cell.

The concept of 1. transformation: it is the process that the target gene enters the recipient cell and remains stably expressed in the recipient cell.

2. Common conversion methods:

Introducing the target gene into plant cells: Agrobacterium transformation is the most commonly used method, followed by particle bombardment and pollen tube pathway method.

Introducing the target gene into animal cells: the most commonly used method is microinjection technology. The recipient cells of this method are mostly fertilized eggs.

Introducing the target gene into microbial cells: Prokaryotes are used as recipient cells because of their fast reproduction, single cells and relatively little genetic material. The most commonly used prokaryotic cell is Escherichia coli. The transformation method is to first treat cells with Ca2+ to become competent cells, and then dissolve the recombinant expression vector DNA molecules in the buffer solution and mix them with competent cells, so as to promote the competent cells to absorb DNA molecules at a certain temperature and complete the transformation process.

3. After the recombinant cells are introduced into recipient cells, the selection of recipient cells containing gene expression vectors is based on whether the marker gene is expressed.

Step 4: Detection and expression of the target gene.

1. First, it is necessary to use DNA molecular hybridization technology to detect whether the target gene is inserted into the chromosomal DNA of transgenic organisms.

2. Secondly, it is necessary to detect whether the target gene has a transcribed mRNA by hybridizing the labeled target gene with the mRNA as a probe.

3. Finally, protein was extracted from transgenic organisms and hybridized with corresponding antibodies to detect whether the target gene was translated into protein.

4. Sometimes it is necessary to identify the biological level of an individual. For example, whether transgenic insect-resistant plants have insect-resistant characteristics.

(C) the application of genetic engineering

1. Plant genetic engineering: Plants have insect resistance, disease resistance and reversibility, and the quality of plants is improved through transgenic technology.

2. Animal genetic engineering: improve the growth rate of animals, improve the quality of livestock products, and use transgenic animals to produce drugs.

3. Gene therapy: normal foreign genes are introduced into patients to make gene expression products play a role.

The concept of protein project.

Protein Project refers to transforming the existing protein or manufacturing a new protein to meet the needs of human production and life according to the structural laws of protein molecules and the relationship between their biological functions. (In principle, genetic engineering can only produce protein that already exists in nature)

Transcription translation

Topic 2 Cell Engineering

(A) plant cell engineering

1. theoretical basis (principle): cell totipotency

The difficulty of totipotency expression: fertilized egg > germ cell > stem cell > somatic cell; Plant cells > animal cells

2. Plant tissue culture technology

(1) process: in vitro plant organs, tissues or cells → callus → test-tube seedlings → plants.

(2) Uses: micropropagation, crop detoxification, artificial seed production, haploid breeding and industrial production of cell products.

(3) Status: Cultivating new plant varieties is the last process of cultivating transgenic plants and plant somatic hybridization.

3. Plant somatic hybridization technology

(1) process:

(2) Methods of inducing fusion: Physical methods include centrifugation, vibration and electrical stimulation. Chemical methods generally use polyethylene glycol (PEG) as inducer.

(3) Significance: Overcome the obstacle of distant hybridization incompatibility.

(2) Animal cell engineering

1. Animal cell culture

(1) Concept: Animal cell culture is to take out relevant tissues from animals, disperse them into single cells, and then put them into a suitable culture medium to allow these cells to grow and reproduce.

(2) The process of animal cell culture: taking animal tissue blocks (organs or tissues of animal embryos or young animals) → slicing → treating with trypsin or collagenase and dispersing into single cells → making cell suspension → transferring into a culture bottle for primary culture → treating cells covered on the bottle wall with trypsin or collagenase and dispersing into single cells, and continuing subculturing.

(3) Cell adhesion and contact inhibition: Cells dispersed in the suspension quickly adhere to the bottle wall, which is called cell adhesion. The number of cells is increasing. When adherent cells divide and grow to the surface and inhibit each other, cells will stop dividing and proliferating. This phenomenon is called cell contact inhibition.

(4) Animal cell culture needs to meet the following conditions

① Sterile and nontoxic environment: the culture solution should be sterilized. Usually, a certain amount of antibiotics should be added to the culture solution to prevent pollution in the culture process. In addition, the culture medium should be changed regularly to prevent the accumulation of metabolites from harming the cells themselves.

② Nutrition: Synthetic medium components: sugar, amino acids, growth promoting factors, inorganic salts, trace elements, etc. It is usually necessary to add natural ingredients such as serum and plasma.

③ Temperature: Suitable temperature: 36.5℃ 0.5℃ for mammals; PH value: 7.2~7.4.

④ Gas environment: 95% air +5% CO2. O2 is necessary for cell metabolism, and the main function of CO2 is to maintain the pH value of the culture medium.

(5) Application of animal cell culture technology: preparation of virus vaccine, preparation of monoclonal antibody, detection of toxic substances, and culture of various cells for medical research.

2. Animal somatic cell nuclear transfer technology and cloned animals

(1) Mammalian nuclear transfer can be divided into embryo nuclear transfer (easy) and somatic nuclear transfer (difficult).

(2) The reason for choosing enucleated oocytes (mother cells) is that oocytes (mother cells) are relatively large and easy to operate; Egg (mother) cells are rich in cytoplasm and nutrition.

(3) The general process of somatic cell nuclear transfer is: (right)

Nuclear transplantation

embryo transplantation

(4) Application of somatic cell nuclear transfer technology:

① Accelerate the process of genetic improvement of livestock and promote the breeding of excellent herds; (2) Protecting endangered species and increasing the number of survivors;

③ Producing precious medical protein; ④ As a xenograft donor;

⑤ Used for tissue and organ transplantation.

(5) Problems in somatic cell nuclear transfer technology:

Cloned animals have health problems and show genetic and physiological defects.

3. Animal cell fusion

(1) Animal cell fusion, also called cell hybridization, refers to the process in which two or more animal cells combine to form one cell. Monocytes with original genetic information of two or more cells formed after fusion are called hybrid cells.

(2) The principle of animal cell fusion is basically the same as that of plant protoplast fusion, and the method of inducing animal cell fusion is similar to that of plant protoplast fusion. Commonly used inducing factors are polyethylene glycol, inactivated virus and electrical stimulation.

(3) Significance of animal cell fusion: It overcomes the incompatibility of distant hybridization and becomes an important means to study cytogenetics, cellular immunity, tumor and cultivation of new biological varieties.

(4) Comparison between animal cell fusion and plant somatic hybridization:

The principle, methods, induction methods and uses of cell fusion are compared.

Cell membrane fluidity of plant somatic hybridization induces protoplast fusion, after cell wall removal, centrifugation, electrical stimulation and vibration, and the incompatibility of distant hybridization is overcome with polyethylene glycol and other reagents to obtain hybrid plants.

The fluidity of animal cell fusion cell membrane disperses cells and induces cell fusion. In addition to plant cell hybridization, inactivated virus was added to induce the preparation of monoclonal antibodies.

4. Monoclonal antibody

Antibody (1): A B lymphocyte secretes only one specific antibody. The antibody isolated from serum has low yield, low purity and poor specificity.

(2) Preparation process of monoclonal antibody

(3) The characteristics of hybridoma cells: they can not only multiply in large quantities, but also produce specific antibodies.

(4) Advantages of monoclonal antibodies: strong specificity, high sensitivity and mass preparation.

(5) the role of monoclonal antibodies:

① As a diagnostic reagent, it has the advantages of accuracy, high efficiency, simplicity and rapidity by accurately identifying the subtle differences of various antigen substances and specifically binding with some antigens.

② Used to treat diseases and carry drugs: mainly used to treat cancer, and can be made into "biological missiles", and a small amount can also be used to treat other diseases.

Theme 3 Embryonic Engineering

(A) the basic process of animal embryo development

1. Embryo engineering refers to various micromanipulation and processing technologies for early embryos or gametes of animals, such as embryo transfer, in vitro fertilization, embryo segmentation, embryonic stem cell culture and so on. The embryos obtained after treatment need to be transplanted into female animals to produce offspring to meet the various needs of human beings.

2. The basic process of animal embryo development.

(1) The fertilization site is the upper tubal segment of the mother.

(2) Cleavage period: Features: cell mitosis, the number of cells is increasing, but the overall volume of the embryo does not increase, or slightly decreases.

(3) Mulberry: Features: When the number of embryonic cells reaches about 32, the embryo forms a dense cell mass that looks like a mulberry. This is a kind of totipotent cell.

(4) Blastocyst: Features: Cells begin to differentiate (the totipotency of cells is still relatively high in this period). The larger cells gathered at one end of the embryo are called inner cell masses, which will develop into various tissues of the fetus in the future. The cavity in the middle is called blastocyst cavity.

(5) gastrula: Features: It has the differentiation of three germ layers, with blastocyst cavity and gastrula cavity.

(2) Embryonic stem cells

1. Mammalian embryonic stem cells (ES or EK cells for short) are derived from early embryos or isolated from primitive gonads.

2. It has the characteristics of embryonic cells, with small volume, large nucleus and obvious nucleoli; Functionally, it has developmental totipotency and can differentiate into any kind of tissue cells of adult animals. In addition, under the condition of in vitro culture, it can not differentiate and proliferate, can be frozen and preserved, and can also be genetically modified.

3. The main uses of embryonic stem cells are:

① It can be used to study the law of individual development and development of mammals;

② It is an ideal material for studying cell differentiation in vitro. Adding taurine and other chemicals to the culture medium can induce es cells to differentiate into different types of tissue cells, which provides an effective means to reveal the mechanism of cell differentiation and apoptosis.

③ It can be used to treat some chronic human diseases, such as Parkinson's syndrome and juvenile diabetes.

④ Using the characteristics of induced differentiation into new tissue cells, transplanted ES cells can repair necrotic or degenerated parts and restore normal functions;

⑤ With the development of tissue engineering technology, by inducing es cells to differentiate in vitro and directionally culturing artificial tissues and organs for organ transplantation, the problems of insufficient donor organs and immune rejection after organ transplantation can be solved.

(C) the application of embryo engineering

1. In vitro fertilization and early embryo culture

(1) collection and culture of oocytes;

Main method: Use gonadotropin to make it excrete more eggs. Then, the egg is washed out from the fallopian tube and directly fertilized with capacitated sperm in vitro. The second method: collecting oocytes from the ovaries of newly slaughtered female animals; The third method is to directly suck oocytes from the ovaries of living animals through ultrasonic detectors and laparoscopy. The collected oocytes must be artificially cultured and matured in vitro before they can be fertilized with capacitated sperm.

(2) Sperm collection and capacitation: Sperm should be capacitated before in vitro fertilization.

(3) Fertilization: capacitated sperm and cultured mature egg cells complete the fertilization process in capacitated fluid or special semen.

(4) Early embryo culture: After sperm and eggs are fertilized in vitro, the fertilized eggs are transferred to the development culture solution to continue the culture, so as to check the fertilization status and the developmental ability of the fertilized eggs. The composition of culture medium is complex. Besides some inorganic salts and organic salts, vitamins, hormones, amino acids, nucleotides and other nutrients, as well as serum and other substances need to be added. When the embryo develops to a suitable stage, it can be taken out, transplanted to the recipient or frozen. Different animals have different embryo transfer times. Cattle and sheep can only be transplanted when they are cultured to morula or blastocyst stage. Mice, rabbits and other experimental animals can be transplanted early, and human in vitro fertilized embryos can be transplanted at 8~ 16 cell stage. )

2. Embryo transfer

(1) Embryo transfer refers to the technology of transferring the early embryos of female animals, or embryos obtained by in vitro fertilization, into other female animals of the same species with the same physiological state, so that they can continue to develop into new individuals. Among them, the individual who provides the embryo is called the "donor" and the individual who receives the embryo is called the "recipient". (The donor is an excellent breed, and the female animal as the recipient should be an ordinary or large original breed. )

Current situation: Any embryo produced by embryo engineering technology, such as transgenic, nuclear transfer or in vitro fertilization, must go through embryo transfer technology to obtain offspring, which is the last "process" of embryo engineering.

(2) The significance of embryo transfer: it greatly shortens the reproductive cycle of the donor itself and gives full play to the reproductive ability of excellent female individuals.

(3) Physiological basis: ① After an animal is in estrus and ovulates, the physiological changes of the donor and recipient reproductive organs of the same animal are the same. This provides the same physiological environment for the donor embryo to move into the recipient.

② Early embryos are in a free state for a certain period of time. This makes it possible to collect embryos.

③ The recipient has no immune rejection to foreign embryos transplanted into uterus. This provides the possibility for the survival of embryos in the recipient.

④ The donor embryo can establish normal physiological and histological connection with the recipient uterus, but the genetic characteristics of the donor embryo are not affected during pregnancy.

(4) The basic procedures mainly include:

① Selection and treatment of donors and recipients. Select donors with excellent genetic characteristics and production performance, and recipients with normal physical health and reproductive ability. Donors and recipients are the same species. Hormone synchronization and gonadotropin superovulation in donor cows.

② Breeding or artificial insemination.

③ Collection, examination, culture or preservation of embryos. On the 7th day after mating or insemination, the embryo in the uterus of the donor cow is washed out with a special egg washer (also called egg washing). Check the quality of the embryo. At this time, the embryo should develop to the stage of mulberry or blastocyst. Transplanted directly into the recipient or stored in-196℃ liquid nitrogen.

④ Embryo transfer.

⑤ Post-transplant examination. Check whether the recipient cow is pregnant.

3. Embryo segmentation

(1) Concept: It refers to the technology of cutting early embryos into 2 and 4 equal parts by mechanical means, and obtaining identical twins or multiple births through transplantation.

(2) Significance: The offspring from the same embryo have the same genetic material and belong to asexual reproduction.

(3) Materials: Morula or blastocyst with good development and normal morphology. During the development from morula to blastocyst, cells begin to differentiate, but their totipotency is still high, which can also be used for embryo segmentation. )

(4) Operation process: When the blastocyst stage embryo divides, the inner cell mass should be equally divided, otherwise it will affect the recovery and further development of the divided embryo.

Topic 4 Safety and Ethical Issues of Biotechnology

(A) the safety of genetically modified organisms debate:

(1) Gene biology and food safety;

Objection: Oppose "substantial equivalence", lag effect, new allergens and changes in nutritional components.

Positive views: safety evaluation, scientists' responsible attitude, no evidence.

(2) Genetically modified organisms and biosafety: impact on biodiversity

The opposite view: spreading out of the planting area to become a wild species, becoming an alien invasive species, recombining harmful pathogens and becoming super weeds may cause "genetic pollution"

Positive views: limited vitality, reproductive isolation, limited pollen transmission distance and limited pollen survival time.

(3) Genetically modified organisms and environmental safety: impact on ecosystem stability.

Objection: Breaking species boundaries, secondary pollution, harmful pathogenic microorganisms and recombination of toxic protein may all enter the human body through the food chain.

Positive view: do not change the original classification state of organisms, reduce the use of pesticides, and protect the farmland soil environment.

(b) Ethical issues in biotechnology

(1) Human cloning: There are two different views, and most people hold a negative attitude.

Negative reasons: human cloning is a serious violation of human ethics and an abuse of cloning technology; Human cloning has impacted the existing traditional ethical concepts such as marriage, family and sexual relations. Human cloning is a person whose psychological and social status is not perfect because of artificial creation.

Positive reasons: Technical problems can be solved by embryo grading, gene diagnosis and chromosome examination. Immature technology can only be matured through practice.

China government's attitude: reproductive cloning is prohibited and therapeutic cloning is not opposed. Four No Principles: Disapproving, disallowing, supporting and not accepting any experiment of reproductive cloning of human beings.

(2) IVF: There are two differences between the two purposes of IVF. Most people agree with different views.

Negative reasons: treating IVF as a human body parts factory is a disrespect for life; Early life also has the right to live. Abandoning or killing redundant embryos is tantamount to "murder".

Positive reasons: It solves the problem of infertility, provides the best and fastest method for treating patients with bone marrow hematopoietic stem cells, and will not cause damage to IVF.

(3) Genetic ID card:

Reasons for denial: the disclosure of personal genetic information leads to genetic discrimination, which will inevitably lead to serious consequences such as genetic unemployment, personal marriage difficulties and interpersonal alienation.

Positive reasons: Through genetic testing, preventive measures can be taken as soon as possible and timely treatment can be carried out to save patients' lives.

(3) Biological weapons

(1) Species: pathogenic bacteria, viruses, biochemical agents, pathogenic bacteria after gene recombination.

(2) Mode of transmission: inhalation, ingestion, contact with infected articles, bite by infected insects, etc.

(3) Features: strong pathogenicity, infectivity in most cases, multiple routes of infection, wide pollution area, long incubation period, difficulty in finding, long harm time, etc.

(4) The Biological Weapons Convention and the attitude of the Government of China.