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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5397_Библиотеки_им_академика_М_И_Перельмана
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A. Mukherjee et al.
9.4.1 Gene Transfer-Based Transgenesis
The process of drug discovery often begins with the identication and isolation of
specic genes that encode proteins with potential therapeutic applications. Once a
gene is isolated, researchers can perform invitro transcription to generate mRNA
and, in turn, translate this mRNA into a cell-free system to produce a small amount
of the corresponding protein (Spreaco etal. 2020). While this approach is useful
for certain biochemical studies, it has limitations when it comes to examining the
protein’s biological activity invivo or guring out its three-dimensional structure
through techniques such as crystallization (Agu etal. 2023).
To effectively decode the gene and translate it into a functional protein, the gene
needs to be inserted into the cells. Cells naturally possess the necessary machinery
for transcription and translation. However, the plasma membranes of several cell
kinds serve as a selective barrier, controlling the uptake of compounds. Some molecules can enter cells through pores, which may be controlled in an open or closed
manner (Zhang etal. 2019). Alternatively, specic carriers facilitate the transport of
molecules into the cell. In some cases, molecules recognize external receptors of the
plasma membrane, forming complexes that trigger endocytosis (Fig.9.1). This process involves the absorption of the intricate and surrounding membrane
(Houdebine 2003).
DNA, being a large, negatively charged molecule, is not able to pass the plasma
membrane on its own. This serves as a protective mechanism for cells against foreign DNA (Houdebine 2003). However, to introduce DNA into cells for various
studies, techniques collectively known as transfection are employed. Transfection
Fig. 9.1 Gene transfer to embryonic stem cells by microinjection technique
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involves different methods to force DNA entry into cells and reach their nucleus. It
is important to note that transfection differs from cell infection (Houdebine 2003),
which is a process involving various mechanisms employed by viruses to deliver
their genomes into host cells. These insights into cellular processes and gene transfer techniques are crucial in the drug discovery process, allowing researchers to
study and manipulate genes for therapeutic purposes.
Several physiochemical phenomena facilitate gene transfer into cells, including
cell fusion, transfection, DNA microinjection, viral vectors, electroporation, and
vectors with particular ligands. Each method has its unique advantages and applications in the context of drug discovery.
9.4.2 Vector-Based Transgenesis
In vivo gene transfer inserts a gene via a vector into an organism, allowing researchers to investigate biological functions. However, the ultimate goal is gene therapy,
which involves delivering genetic information to target cells to replace defective
functions or introduce additional functions. Ex vivo gene therapy involves extracting target cells, inserting desired genes, and returning them to the patient, but has
limited results.
In vivo gene therapy faces challenges due to the lack of an efcient, non-toxic
gene delivery system (Fig.9.2). Viruses can be used to transduce cells by replacing
non-essential viral genes with therapeutic genes. Despite triggering a host immune
response, viruses have evolved countermeasures to enter and multiply within the
intended cells. In 1973, the rst attempt was made to employ viruses as medicinal
agents, but replication-competent viruses remain a research eld (Bouard etal. 2009).
Fig. 9.2 Vector-based transgenesis
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9.5 Transgenesis: Target Identication
A transgene is a gene that has been spontaneously transmitted from one creature to
another or has been created by one of several genetic engineering methods. A procedure called transgenesis, which involves the insertion of a transgene, can alter an
organism’s phenotype. A transgene is a section of DNA that has been extracted from
one organism and inserted into another, carrying a gene sequence. This non-native
DNA segment may change the transgenic organism’s genetic code from functioning
normally, or it may preserve the transgenic organism’s capacity to create RNA or
protein. Usually, the DNA is integrated into the germ line of the organism (Gordon
and Ruddle 1981). Gene therapy and transgenesis share the similarity of transforming cells for a certain objective, but their goals are very different: Gene therapy aims
to x a disease in cells, while transgenesis seeks to generate a genetically transformed organism by inserting a specic transgene into each cell and changing its
genome (Moody etal. 1981).
Mouse molecular genetics underwent a revolution in the 1980s. The rst transgenic mouse was produced by injecting cloned DNA into fertilized mouse eggs
(Gordon etal. 1980). Investigators quickly reported germline transfer of transgenes
via nuclear injection of DNA (Costantini and Lacy 1981). Transgenic mice have
now been employed in a frenzy of research that has not stopped examining a variety
of topics, such as tissue-specic gene expression, oncogenesis, and developmental
alterations (Neidhardt etal. 2000).
9.5.1 Transgenic Target Identication
There are several methods for nding novel possible pharmacological targets
(Debouck and Goodfellow 1999). The genetic approach and the genomic approach
are two conceptually distinct methods (Schmid etal. 2021). The genetic method
examines the differences in mRNA expression between, say, healthy and diseased
tissues or the alterations in gene expression that occur when a substance is introduced to a cell culture medium or administered to an animal (Alberts 2017). These
days, a lot of organizations employ proteomics and microarray technologies to nd
variations in gene expression (Lockhart and Winzeler 2000).
9.5.2 Meaning oftheTarget Identication
The process of gathering data for and against a target is known as target identication and validation. To gather enough information, medication developers usually
spend 12–18months (about 1 and a half years) either performing original research
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or replicating existing studies in the literature (Békés etal. 2022). In the absence of
a small-molecule tool, genetically modied animals, neutralizing antibodies, and
small-interfering ribonucleic acid (siRNA) can be used as research tools for validation; nevertheless, their capacity to understand the pharmacodynamics of the drugtarget interaction is often restricted. (MacDonald etal. 1993).
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9.5.3 Difference andImportance Between Target
andIndicators
There are two primary reasons why drugs fail in clinical settings: they are unsafe
or they do not function. Therefore, target selection and validation are among the
most crucial phases in the development of a novel medication. A target is a general
phrase that may be used to describe a variety of biological things, such as proteins,
genes, and RNA.A good target must be “druggable,” safe, suit clinical and commercial goals, and be effective. Whether a tiny molecule or a bigger biological
one, a “druggable” target is reachable by the possible therapeutic chemical and,
upon binding, causes a biological response that can be evaluated both invitro and
in vivo. It is now established that while antibodies are effective in obstructing
protein/protein interactions, other target classes—such as GPCRs, which are connected to proteins—are more amenable to the development of small-molecule
drugs. A well-dened and validated objective allows for more trust in the connection between the target and the illness and enables us to investigate whether changing the target will have side effects based on a mechanism (Henning and
Beste 2002).
9.5.4 Aim ofTransgenic Target Identication
While homologous integrations do happen and transfer DNA to a specic site in
the genome, random integrations of imported DNA predominate. The early cases
of gene targeting required stringent screening for the rare homologous occurrences, which occurred at around 10−3–10−5 of total integrations, regardless of
whether the locus was “natural” or articially produced (Smithies etal. 1985; Lin
etal. 1985). Since then, several ES cell examples have typically produced targeted
frequencies of 10−2 or greater for the overall number of integrations. The percentage of targeted clones among selected clones can be increased by including enrichment techniques in the selection process (Mansour etal. 1988). Drug selection is
crucial for isolating targeted clones since a targeting frequency of 10−2 of total
integers corresponds to a 10−5–10−6 absolute targeting frequency of the transfected
cell population.
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9.5.5 Properties ofGood Gene Target
Still, it can be difcult to predict how transgenes, especially targeted ones, would
express themselves. Targeting the lacZ reporter gene to the third exon of hprt was
noteworthy because the targeted clones exhibited varied expressions that depended
on both orientation and cell type (Shaw-white etal. 1993; Maltsev etal. 1994). Like
lacZ targeted to the hprt locus, in this instance, ES clones also showed variable
β-galactosidase staining patterns. In both scenarios for the variegated staining patterns, promoter occlusion effects may be accountable because it has been shown
that nearby promoters might affect one another (Alam and Cook 1990; Barnes etal.
2000). It might be possible to prevent promoter occlusion effects with such a design.
It is challenging to come to a denitive judgment in this regard, nevertheless,
because Northern analysis was used to monitor the expression of bcl-2 rather than
expression within individual cells.
9.5.6 Method andTypes ofTarget Identication
Target validation is an ongoing, crucial procedure that begins at the beginning of the
discovery phase. Various steps of this procedure need the application of different
validation techniques. Cultured cells and bioinformatics technologies can be utilized in the very early stages when choices must be made among several targets.
Before signicant resources are committed to the possible target, there is an urgent
need for invivo TV data. Although a lot of work is being done to nd quick, dependable, and universal methods for invivo validation, transgenic animals are the only
ones that have proven effective so far on a variety of targets. A target and possible
medication combination cannot be considered fully “validated” until the novel medicine has been evaluated in clinical trials. The creation of genetically altered animals
that either overexpress (gene addition) or lack the target (knockout animals) is a
typical necessity in the TV phase. These animals offer functional invivo data—
which are frequently lacking—on a possible target. These days, varying degrees of
complexity can be used to modify the target gene’s expression in genetically engineered animals. One that overexpresses the transgene either broadly or selectively
in a particular tissue is the fastest-growing transgenic animal. Nowadays, a wide
variety of tissue-specic promoters have been used to restrict transgene expression
in different cell types. (Morimoto et al. 2001; Burton et al. 1991; Field 1988).
Several mechanisms exist for controlling transgenic expression using nonphysiological inducers. The phosphoenolpyruvate carboxykinase promoter and the
metallothionein promoter were the rst inducible systems employed in transgenic
animals. There were issues with background expression levels, and these systems
were hard to control. With several successful trials under its belt, the tetracycline
system is currently the most popular method for regulating transgenes. Although
technologies for articial dimerization of molecules and the ecdysone system have
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also been created, it is yet unclear how broadly these systems may be used to regulate transgenes. Furthermore, gene activity has been regulated via fusion proteins
that link an effector protein (such as Cre) to a steroid receptor’s ligand-binding
domain (Lim etal. 1990; Ewald etal. 1996; Furth etal. 1994; Perea etal. 2001; No
etal. 1996; Amara etal. 1997).
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9.5.7 Steps ofTransgenic Target Identication
DNA Preparation andPurication
DNA Construct/Fragment Structure
DNA microinjection appeared to be one of the only gene transfer techniques where
fragment size was unaffected when compared to other techniques (especially when
it came to retro-viral packaging); however, the initial limitations on fragment size
were imposed by the cloning vectors cosmid (45kb) and plasmid (12kb). Large
chromosomal segments and vectors such as the yeast articial chromosome (YAC),
P1, and bacterial articial chromosome (BAC) can now be cloned in addition to
bigger stable constructions.
Preparation ofDNA forMicroinjection
(a) Purication of DNA
• After verifying correct cloning and conrming the nucleotide sequence, the
DNA fragment undergoes purication steps.
• The goal is to ensure that the DNA fragment is free of nicks or strand breaks
and is as pure as possible.
(b) Use of High-Quality Reagents
• High-quality reagents are emphasized for the nal purication steps.
• Solutions are made from tissue culture-grade, 18MW water.
(c) Water Purication Systems
• Commercial water systems are utilized, combining reverse osmosis and distillation combined with ultraltration or deionization.
• Water systems from various manufacturers, such as Millipore’s Milli-Q,
Barnstead’s NanoPure, and Corning’s Mega-Pure, are mentioned.
(d) Filtration of Solutions
• Solutions are processed using a 0.45- or 0.2-mm lter after preparation.
• Host Strain for Plasmid Replication
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• It is possible to reproduce plasmid DNA in any common Escherichia coli host.
• CPLK-17, the DH5 methylase-defective strain, is recommended for maintaining the methylation state of the gene construct during cloning.
(e) Transgene Rescue and Analysis
• The CPLK-17 strain allows transgene rescue for toxicology applications and
the analysis of anking regions of the chromosomal insertion.
• This facilitates the localization of insertional mutants.
(f) Cloning and Gene Construct Assembly
• Techniques for basic cloning and gene construct assembly are referenced.
• Large-scale DNA preparation is mentioned, with references to welldocumented methods.
(g) Linearization and Microinjection Preparation
• Before microinjection, the plasmid DNA is puried by isolating supercoiled
DNA from a cesium chloride (CsCl) gradient.
• Using restriction enzyme digestion, the gene fragment is then separated
from the vector sequences.
(h) Minimization of Flanking Vector Sequences
• The passage highlights the routine minimization of the amount of anking
vector sequences for injection fragments.
• Vector DNA sequences can inuence the function of foreign genes after
chromosomal integration.
(i) Construct Design
• The construct should be designed to facilitate easy separation of DNA bands
migrated on agarose gels.
Superovulation, Egg Culture, andHarvest
(a) Popular Mouse Hybrid for DNA Microinjection
• Several hybrids are commonly used for DNA microinjection.
• The C57BL/6 SJL (B6SJL) F1 hybrid is specically mentioned as being
efcient in generating transgenic mice.
• References are made to studies supporting the efciency of this hybrid.
(a) Preference for C57BL/6 Inbred Strain in Parental Stocks
• The C57BL/6 inbred strain is one of the parental stocks in most hybrids used
for microinjection.
• This choice is based on favorable genetic and embryological characteristics
associated with the C57BL/6 strain.
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(b) Advantages of Hybrid Mice
• One of the main benets of employing hybrid mice for microinjection is
their accentuated hybrid vigor.
• Hybrid vigor not only imparts desirable reproductive characteristics but also
enhances egg quality, leading to favorable microinjection characteristics.
(c) Considerations for Genetic Background Uniformity
• There is a cautionary note about the need to be concerned with the uniformity of the genetic background in which the transgene will be functioning.
• This consideration is particularly important for experimental designs requiring large populations or many generations of transgenic mice.
Microinjection Needles andSlides
(a) Slide Option
• Cut-out or depression slides can hold eggs and DNA.For cut-out slides, an
equivalent area is removed from a at glass slide instead of creating a depression in the center.
(b) Cut-Out Slides and Disposable Coverslips
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• Cut-out slides use disposable glass coverslips for holding microdrops overlaid with oil.
• The coverslips are attached to the bottom of the cut-out slide using a ring of
parafn, positioned outside the periphery of the hole.
(c) Advantages of Cut-Out Slides
• Cut-out slides offer advantages over depression slides.
• Coverslips are disposable.
• There is less light refraction.
(d) Conguration Details
• To prevent the cover slip from contacting surfaces, on the bottom of the slide
at both ends, 1–2mm blocks or risers are employed.
(e) Needle Manipulation Considerations
• Due to the conguration of cut-out slides and the need for a greater needle
angle when working with dishes on a microscope, needle tips must be bent
at approximately a 30° angle prior to microinjection.
A HEPES-buffered medium is utilized for microinjection; examples include
modied BMOC-3 plus HEPES (recipes in Table9.1) supplemented with cytochalasin B (5mg/mL). The egg will probably lyse quickly if the injection causes the
egg membrane to be pulled into the cytoplasm. During microinjection, cytochalasin
B stiffens the membranes and aids in preventing egg lysis. Conversely, 7% (v/v)
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ethanol produces a comparable result (P. Hoppe, private correspondence 1991).
Another method for stiffening egg membranes is to utilize a stage cooler (e.g., KT
Model 5000, Technology, Inc., Whitehouse Station, NJ) to reduce the slide temperature to 10°C.As long as the eggs are returned to 37.5°C within 45min, neither the
proportion of transgenic mice produced nor egg survival will be affected by this.
Microinjection Equipment
It is frequently employed, and a choice must be considered before making one. The
brand or design of the microscope may have an impact on the nal differential interference contrast (DIC) magnication, and prior training or personal experiences
often determine the ideal eld diameter. DIC is often only needed at 200¥ microinjection magnication. Although it offers a sizable working eld for sorting eggs and
priming pipettes, low-power magnication does not need DIC optics. It is thus recommended to test the nal setup and any required changes (such as light lament,
lens, diaphragms, and focusing lenses) prior to purchase, ideally when the manufacturer’s representative and a small number of eggs are accessible at the same time.
The representative may congure the DIC in a way that is deemed “optimal,” but
this does not guarantee the nest pronuclear pictures (Rusconi 1991, 1996).
A. Mukherjee et al.
9.5.8 Problem inTransgenic Target Identication
Since cloning has made it easier to add and replace genes, animal transgenesis has
a signicant inuence on human health. Agricultural animal species’ wholegenome sequencing provides more genes for study and helps create advantageous
animal lines. Although transgenic animals are useful for researching human illnesses and creating therapeutic proteins, moral dilemmas still exist. The use of
transgenesis to improve animal production presents difculties in assessing the
potential medical effects. Although gene transfer is progressing, barriers such as
high costs for transgenic farm founders and delayed trait transmission remain,
meaning transgenes must provide signicant benets before they can be used in
practice (Houdebine 2005).
9.6 Transgenesis: Target Validation
9.6.1 Validation Method
That is, by comparing computations with a collection of experimental data, we may
determine how condent we are in a code’s capacity to anticipate results for a particular application (Trucano et al. 2006). Within the context of studies on the
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validity of aptitude tests for people evaluation, the notion of situational specicity
has taken on rather varied roles. The validity coefcients were shown to differ
greatly when standardized aptitude tests were rst associated with performance on
seemingly identical occupations in industrial validation studies (Anastasi 1986).
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9.6.2 Target Validation Method
Like target identication, target validation veries that the interaction of the target
has the potential to be therapeutically benecial. A target will not be developed
further in the drug development process if it cannot be veried. Samuel Gandy,
professor in the departments of neurology and psychiatry and associate director of
the Alzheimer’s Disease Research Center at Mount Sinai, and Reisa Sperling, professor of neurology at Harvard Medical School and director of the Center for
Alzheimer’s Research and Treatment, talked about two opportunities: early validation of targets and improved biomarkers. Eli Lilly and Company’s Chief Scientic
Ofcer for Tailored Therapeutics-Neuroscience, Kalpana Merchant, furthermore,
provided a portfolio evaluation tool that outlined certain parameters for target validation and qualication. The crucial rst phase in the development of a new medication is target validation, which normally takes two to six months to nish. In order
to prove that the drug’s actions on the target can provide therapeutic advantages
within an acceptable safety window, a variety of procedures must be applied. The
likelihood of success in clinical trials is greatly increased when early and thorough
target validation is implemented, as it helps to establish a clear correlation between
target alteration and disease effectiveness. A target moves on to the hit identication
phase of the research after it reaches a satisfactory degree of validation and illness
correlation.
9.6.3 Animal Model Validity
A scientic technique to increase a model’s credibility is to validate it or assess its
consistency and plausibility. Validity refers to the extent to which a test’s score or
measure aligns with the attribute it is intended to evaluate. It is the interpretation of
the data derived from this model that is validated, not the animal model. In that
regard, validity is a crucial factor to consider when assessing animal models. There
is not one animal model that works for everything or in every circumstance. Because
validity is limited to a particular application of the model, it must constantly be
available for debate and revision (Kaplan and Saccuzzo 2001; Silva 1993).
Regarding the relative importance of the various validity criteria in the model review
process, there is no universal agreement. We maintain that a model’s construct
validity, external validity (i.e., gen- realizability), predictive validity, and reliability
and replicability (internal validity) should all be taken into account throughout the
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