The methods used to introduce foreign DNA into animal embryos
Introduction of foreign DNA to mice was carried out by different methods: 1) using retroviral vectors that infect embryonic cells at early stages of development before implantation of the embryo into the female recipient; 2) by microinjection into the enlarged sperm nucleus (male pronucleus) of a fertilized egg; 3) introduction of genetically modified embryonic stem cells into a pre-implanted embryo at early stages of development.
Use of retroviral vectors. The advantage of the method based on the use of retroviral vectors, over other methods of transgenesis, is its effectiveness. However, the size of the insert in this case is limited to 8 bp, so that the transgene may be devoid of adjacent regulatory sequences necessary for its expression.
The use of retroviral vectors has another major drawback. Although these vectors are created so that they are replication defective, the genome of the retrovirus strain (helper virus), which is necessary for obtaining a large amount of vector DNA. can get into the same core as the transgene. Despite all the measures taken, retrovirus assistants can replicate in the body of a transgenic animal, which is completely unacceptable if these animals are intended to be used as food or as a tool for obtaining a commercial product. And since there are alternative methods of transgenesis, retroviral vectors are rarely used to create transgenic animals of commercial value.
Method of microinjection of DNA. At present, the method of microinjection of DNA is most often used to create transgenic mice. It consists in the following. Work begins with the stimulation of hyperovulation in donor females to increase the number of oocytes in which foreign DNA will be injected. First, the females are injected with the serum of the pregnant mare, and after about 48 hours - human chorionic gonadotropin. As a result of hyperovulation, approximately 35 eggs are formed instead of the usual 5-10 eggs. Then cross with males of females with hyperovulation, then kill them, wash eggs from fertilized eggs, and immediately inject DNA into fertilized eggs. Often the introduced transgenic construct is in a linear form and does not contain prokaryotic vector sequences.
In mammals, after penetration of the spermatozoon into the egg cell, the spermine nucleus (male pronucleus) and the nucleus of the oocyte exist separately. After the latter finishes the mitotic division and becomes a female pronucleus, a fusion of nuclei (karyogamia) can occur. The male pronucleus is usually much larger than the female pronucleus, it is easy to localize with a sectional microscope and insert foreign DNA into it. At the same time, the egg can be moved, oriented and fixed during the microinjection. Experienced experimenters can inoculate several hundred eggs a day.
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After the introduction of DNA from 25 to 40 eggs are implanted microsurgically into the "surrogate" mother, who is induced by a false pregnancy by crossing with a vasectomized male. In mice, mating is the only known way of preparing the uterus for implantation. Since the vasectomized male sperm does not produce, none of the eggs of the "surrogate" mother is fertilized. Embryos develop only from injected eggs, and the mice are born about 3 weeks after implantation.
To identify transgenic animals, DNA is isolated from a small piece of tail and tested for transgene by Southern blot hybridization using the PCR method. To determine whether the transgene is in the cells of the germ line of the animal, the transgenic mouse is crossed with another mouse. Further, it is possible to crossbreed offspring to obtain pure (homozygous) transgenic lines.
The approach described seems at first sight relatively simple, but it requires a clear coordination of the different stages. Even a highly qualified specialist manages to obtain viable transgenic animals at best from only 5% of inoculated oocytes. None of the stages of the experiment is effective at 100%, therefore for microinjection it is necessary to use a large number of fertilized eggs. For example, when producing transgenic mice after DNA injection, only 66% of fertilized eggs survive; mice develop about 25% of implanted eggs, and transgenic of them are only 25%. Thus, from 30 to 50 transgenic mice develop from 1000 implanted fertilized eggs. In addition, the inserted DNA can integrate anywhere in the genome, and often many of its copies are included in one site. And, finally, not all transgenic mice will have the necessary properties. In some organisms, the transgene may not be expressed due to an improper environment of the integration site, and in the body of others, the number of copies of the foreign gene may be too large, which can lead to hyperproduction of the protein and disruption of normal physiological processes. And yet, despite all this, the method of microinjection is used to obtain the lines of mice bearing functional transgenes quite often.
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Use of modified embryonic stem cells. Cells isolated from murine embryos in the blastocyst stage can proliferate in culture, retaining the ability to differentiate into any cell types, including germline cells, when introduced into another embryo at the blastocyst stage. Such cells are called pluripotent embryonic stem cells (ES). ES cells in culture can easily be modified by genetic engineering methods without disturbing their pluripotency. For example, in a particular site of a nonessential gene, a functional transgene can be embedded in their genome. Then you can select the changed cells, cultivate them and use them to produce transgenic animals. This avoids the accidental embedding that is characteristic of the method of microinjection and retroviral vector systems.
When transfecting ES cells in culture with a vector designed for integration into a specific chromosomal site, DNA is randomly embedded in some cells, in others, embedding occurs at the desired site, most ES cells do not integrate at all. The so-called positive-negative selection is used to increase the number of cells of the first type. This strategy consists in the positive selection of cells carrying vector DNA embedded in the desired site, and negative selection of cells with vector DNA that integrated into a random site.
The target site should be in a genomic DNA region that does not encode important proteins, so that the integration of foreign DNA does not affect developmental processes or cellular functions. In addition, it is essential that the incorporation of the transgene does not block the translation of the corresponding region of the genome. The search for such sites is ongoing.
An easier way to identify ES cells carrying the transgene in the desired site is based on the use of PCR. In this case, the DNA vector contains two regions homologous to the target site, one from the side of the transgene and from the side of a cloned bacterial or synthetic (unique) sequence absent in the mouse genome. After transfection of the ES cells with this vector, screen the transfected cells by PCR. One of the PCR primers is complementary to the portion of the cloned bacterial or synthetic (unique) nucleotide sequence of the integrated vector, and the second to the region of the chromosomal DNA adjacent to one of the homologous DNA regions. When the target sequence is inserted into a random site, the expected amplification product will not be formed, and site-specific integration results in a DNA fragment of known size resulting from PCR amplification. In this way, it is possible to identify pools of ES cells containing the transgene in the desired site, and by crossing cells from these pools, get cell lines with a site-specific insert.
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ES cells, in the genome of which the transgene is embedded in the desired site, can be cultured and introduced into the embryo at the stage of the blastocyst, and then implant such embryos into the uterus of pseudobremenal "surrogate" mothers. Mice, in which genetically modified ES cells participated in the formation of germline cells, can give rise to transgenic lines. For this, they must be crossed with individuals of the same line, and then crossed their transgenic offspring. As a result, transgenic mice that are homozygous for the transgene will be obtained.
In principle, approaches to the creation of transgenic animals with "improved functions" and with "loss of functions" are similar. Unfortunately, pluripotent ES cells, similar to those in mice, have not yet been found in cattle, sheep, pigs and chickens, but their search continues.
Transgenic mice can serve as model systems for studying human diseases and test systems for investigating the possibility of synthesis of products of interest to medicine. Using whole animals, it is possible to model the appearance of pathology, and its development. However, the mouse is not a human, although it also belongs to the class of mammals, therefore the data obtained from transgenic models can not always be extrapolated to humans in terms of medical aspects. Nevertheless, in some cases, they allow us to identify the key points of the etiology of a complex disease. Taking into account all this, scientists have developed "mouse" models of such genetic diseases as Alzheimer's, arthritis, muscular dystrophy, tumor formation, hypertension, neurodegenerative disorders, endocrine system dysfunction, cardiovascular diseases and many others.
To create transgenic cows, a modified transgenic mice transplantation scheme using a microinjection of DNA is used. The procedure includes the following main steps: the collection of oocytes from cows slaughtered in a slaughterhouse; maturation of oocytes in vitro; Bovine semen fertilization in vitro; centrifugation of fertilized eggs to concentrate the yolk, which in normal ovules interferes with the visualization of the male pronucleus with the help of a sectional microscope; microinjection of DNA into the male pronucleus; development of embryos in vitro; non-surgical implantation of one embryo to the recipient female during estrus; screening of DNA descendants for the presence of a transgene.
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