Gene transfer is a technique which includes both biological research and technological research. It still remains as an important and active area of research and clinical investigation. Gene therapy strategies are very much useful for tissue engineering by modifying cells directly of an animal providing him a favorable growth. To accomplish this, cells are modified genetically by various methods using genetic transfer vectors. This method is being done by the process of transferring the therapeutic DNA into the nucleus where it is transcribed in parallel with genomic DNA. A variety of non-viral and viral gene transfertherapy vectors have been developed for this method that include plasmids. Not only that, plasmids also combined with liosomes, adenovirus, adeno-associated virus (AAV), retrovirus and lentivirus to make this method happen. The value in using viral vectors, especially adeno-viral vectors, for gene transfer in the vitro is that these vectors are highly efficient in transferring genes. However, by using a relatively modest dose of an adeno-viral vector, an experienced professional investigator can routinely and reproducibly transfer the gene of interest to 100% of the target cells.
Generally, the gene transfer process has been done between animals. It’s not that any random animals but most importantly all animals undergoing this transfer continue to be monitored for the long-term safety and efficacy of all of these new methods. A highly unexpected desirable outcome in these genetic transfer studies has been transgene expression over 10 years albeit at low levels but without detectable toxicity. Previously the success rate of this method was not that good!
However, on the arrivals of World Precision Instruments (WPI’s) Micro-ePORE system, the scenario has got completely changed. These observations are particularly important when considering the fact like the safety aspects of the animals. However, the success rate is getting better by leaps and bounds. In most cases, this process is being done on mice but apart from that, this process is being tried over many other animals and fishes, too.
The DNA or selected gene is introduced by microinjection through a fine glass needle into the male pronucleus – the nucleus provided by the sperm before fusion with the nucleus of the egg. After fertilization of the egg, the manipulated fertilized ovum is transferred into the oviduct of a recipient female or a foster mother that has been induced to act as a recipient. Here are two basic types of microinjection systems. The first is called a pulsed flow system and the second is called a constant flow system.
Figure 1: Microinjection through a
fine glass needle
Intracerebral
Delivery in Complex 3D Arrays is Additional applications of the IMI include
delivery of viral vectors for gene therapy, infusion of neurotrophic factors,
targeted delivery of chemotherapeutic agents, and delivery of antiretroviral
medications.
The described techniques for the introduction of molecular probes in the form of synthetic RNA by rapid repetitive into oocytes (a cell in an ovary which may undergo meiotic division to form an ovum) or early embryos of echinoderms and various invertebrates. Construct assembly is followed by standard kit-based in vitro RNA synthesis, with slight modifications to optimize expression and clean-up. Various types of a basic microinjection procedure are detailed for purple urchin, echinoderms, starfish oocytes, and sand dollar zygotes included for other invertebrate eggs and embryos as well.
RNA export is a poorly understood step in eukaryotic gene expression. Microinjection-based assays allow for the accurate measurement of mRNA nuclear export kinetics. Successful execution of this assay can be aided by implementing certain practices.
Figure 2: Successfully placed DNA or
selected gene in the nucleus of the egg
A network
structure featured with oriented nanohybrid shish-kebab structure was formed.
Composites with improved modulus and strength in parallel with enhanced
toughness were obtained.
Quantitative-microinjection using fluorescence(the property of absorbing light of short wavelength and emitting light of longer wavelength) calibration of streaming microdroplets on a superhydrophobic surface. Superhydrophobic surfaces prevent droplet surface deformation and adhesion. Wide tipped micropipettes create a stream of microdroplets. Streaming creates a large number of varying sized droplets quickly and easily. Fluorescence-volume calibration curves can be created with tracer microdroplets. Calibrated fluorescent tracers allow for quantifiable, cellular-microinjection. Myostatin inhibitor microinjection delayed in vitromaturation and reduced in vitro fertilization and cleavage rates. In the Myostatin inhibitor, the process is also inhibited GDF9 and BMP15 gene expression in cumulus-oocyte complexes.
The histaminergic compounds injected in the cerebellum affect mice’s behavior in the open field. These injections of chlorpheniramine into the cerebellar vermisincreases thelocomotor and exploratory behaviors in the mice. Injections of VUF-8430 into the cerebellar vermis increase locomotor and exploratory behaviors in mice.
The Preparation of retrieved cells to make them suitable for transplantation denotes Cell manipulation.
Recent advances in microfluidic technology for manipulation and analysis of biological cells are Advances in microfluidic technology for the cell analysis in the last decades that were reviewed. Microfluidic platforms exhibited superior spatiotemporal resolution for cell manipulation and monitoring of cellular responses. The commercialization of microfluidic technology has gained traction in biomedical research and diagnostic applications.
Figure 1: Showing the Microfluidic Palette
Digital microfluidics is an important method for cell manipulation. Digital microfluidics (DMF) operating discrete droplets for cell culture, cell sorting, and single-cell analysis. Suspension cell, microorganism, two-dimensional (2D) adherent cell and three-dimensional (3D) cell culture happens on the DMF devices. Cell sorting and concentrating happens within DMF droplets with integrated electrical, optical and magnetic forces. Single-cell culture, stimulation, immunocytochemistry, DNA analysis, mRNA extraction and transcriptome analysis happens with the DMF. Unlimited Genetic Switches for Cell-Type-Specific Manipulation is a new technology to gain independent genetic access to discrete cell types. The multiplexing potential of CRISPR exploited to create infinite genetic switches. A gene-trap system is generally used to discover the lineage-specific genes. The multi-gene intersectional genetics is a vertebrate and invertebrate model.
SponGee is a Genetic Tool for Subcellular and Cell-Specific cGMP Manipulation. The cGMPsometime becomes critical to some of the variety of cellular processes. However, the available tools to interfere with endogenous cGMP lack cellular and subcellular specificity. The SponGee is a genetically encoded chelator of this cyclic. SponGee is a genetically encoded cGMP scavenger. SponGee inhibits cGMP-dependent downstream pathways. SponGee enables cell-specific manipulation of cGMP-dependent processes in vivo. Subcellular targeting confers cell compartment specificity to SponGee.
Figure 2:
Showing Cell Manipulation process
Cell Manipulation Technologies:
Activities of cells are dependent on extracellular niches. The change of the cells is changing quite dynamically. Dynamic substrates are there whose chemical or physical properties can be controlled by an external stimulus. These properties are useful to resolve how cells respond to such dynamic environmental changes. They are useful for cell manipulations, such as heterotypic cell coculturing, cell sheet harvesting, and cell migration induction. We have developed for the first time a dynamic substrate, which is based on the photocleavage reaction of the 2-nitrobenzyl group. One of the biggest advantages of the photochemical approach is its high spatiotemporal resolution. However, there is a lot of room for innovation to improve the switching efficiency and to expand the time window of its usage.
Microinjection is a method that requires a direct injection of DNA into the nucleus of a cell with the aid of a very fine needle. This recombinant DNA absence the genes required for retroviral replication and assembly of viral particles. The target is commonly a living cell but may also include intercellular space. Microinjection being an easy and simple mechanical process, usually including an inverted microscope with an amplification power of around 200x (though sometimes it is performed using a cut-up stereo microscope at 40–50x or a traditional compound upstanding microscope at similar power to an inverted model).
For processes such as pronuclear (relating to either of a pair of gametic nuclei,) or cellular injection the target cell is placed under the microscope and two micromanipulators—one holding the pipette and one holding a micro-capillary needle usually between 0.5 and 5 µm in diameter (larger if injecting stem cells into an embryo)—are used to penetrate the cell membrane and/or the nuclear envelope. In this way, the process can be used to launch a vector into a single cell. In the study of cell biology and viruses, microinjection also can be used as cloning of organisms, and for treating male subfertility through intracytoplasmic sperm injection. In this process the scientists injects DNA molecules directly into male pronuclear. It inserts the manipulated fertilized ovum into the oviduct of a recipient female or fosters mother. This is the most popular technology, commercially available. Here are two basic types of microinjection systems. The first is called a pulsed flow system and the second is called a constant flow system.
History:
Microinjection is a biological process. This procedure began in the early 20th century. Even though the 1970s it was not commonly used. By the 1990s, its use had risen rapidly significantly and it is now considered a common laboratory technique.
Advantages of microinjection:
The frequency of a stable combination of DNA is far better as compared to other methods.
The DNA injected in the process is subjected to less extensive modifications.
The method is effecting in transforming primary cells as well as cells in established cultures.
Mere precise integration of the recombinant gene is a limited copy number can be obtained.
Disadvantages of microinjection:
It’s a costly method.
Skilled personal required.
More useful for animal cells
Knowledge of mating timing, oocyte recovery is essential.
Applications of microinjection:
The process is applicable to plant cells as well as animal cells. But it is quite common for animal cells.
The technique is very useful for producing transgenic animals quickly.
Technique is important for gene transfer to embryonic cells.
Cell manipulation means the preparation of retrieved cells
to make them suitable for transplantation. Experimentation on animal or human
stem cells that alters either their genetic makeup or the biochemical that
those cells produce. Over the past decade (a period of ten years.),
miniaturized devices known as ‘micro total analysis systems’ or ‘lab on a chip’
devices have been repeatedly developed for cell manipulationand
analysis. Soft lithography introduced in the late 1990s has become the most
common method.
Cell manipulation in microfluidics:
Recent advances in the lab-on-a-chip field in association
with microfluidics have been made for new applications and functionalities to
the fields of molecular biology, genetic analysis, and proteomics, enabling the
expansion of the cell biology field. Microfluidics hasdelivered encouraging
tools for growing cell biological research since it has the ability to
definitely control the cellular environment.It is used to easily imitate
heterogeneous cellular environment by multiplexing, and to analyze sub-cellular
information by high-contents screening assays at the single-cell level. Various
cell manipulation techniques in microfluidics have been developed in agreement
with specific objectives and applications. In this review, we inspect the
latest achievements of cell manipulation techniques in
microfluidics by categorizing externally applied forces for manipulation: (i)
optical, (ii) magnetic, (iii) electrical, (iv) mechanical and (v) other
manipulations. We moreover focus on history where the manipulation techniques
originate and also discuss future perspectives with key examples where
available.
Cell manipulation could lead to a new laboratory tool that
will allow scientists to build and move microscopic cells that could lead to
the development of better treatments for disease. Scientists have uncovered how
microscopic cells can be manipulated and studied more clearly in 3D using a
high-intensity infrared light.
The accurate study of cells has earlier been limited by technologies that were only capable of studying cells on a macro scale. This latest development will allow scientists, in the laboratory, to restore the tiny worlds where cells live and in turn understand how they grow and function.
Specific advantages in micro device-based cell manipulation
research include:
Miniaturized devices present enormous opportunities to analyze, characterize, and manipulate cells.
High throughput
Reduction of reagent consumption and the number of cells required,
Tight control of physical and chemical factors,
Tight control of signal gradients
Single-cell manipulation.
Applications of cloning in animal breeding and importantly, in cell biology, blossomed. Hurdles to cell manipulation were overcome and new technologies have evolved.
Microinjection
as a biological procedure began in the early twentieth century, although even
though the 1970s it was not commonly used. So basically Microinjection is the use of a glass micropipette to inject a
liquid substance at a microscopic level. This injection is a proven and
relatively simple method for introducing DNA into worms. Though this process
has a very low success rate but very quickly this process will get the highest
success rate.
DNA
transformation techniques are typically required to co-transformation with a
scoreable or selectable marker gene. Transformation markers that induce a
dominant phenotype allow the transformation of any animal strain as long as the
host’s phenotype does not interfere with the marker-induced phenotype. Other
selectable markers rescue lethal or non-lethal mutations and require the use of
specific mutant strains as transformation hosts. Some mutant rescue
experiments, a co-injected marker may not be necessary but it is usually
advisable as a positive control for transformation.
We
have discovered WPI MICRO-ePORE, as an advanced tool & technique. This
machine can ensure a much better success rate time. WPI MICRO-ePORE, works as
DNA Microinjection has wider benefits. DNA is inserted into animal cells using
microinjection. This machine has a touch screen display, user can adjust
frequency and voltage through the touch screen. This machine can control
injection through foot switch or manually through the touch screen. It has an
adjustable audio continuity tone indicating an active probe Injection counter
to indicate the total number of injections.
Benefits of using WPI
MICRO-ePORE:-
Works with all major inverted microscopes
User adjustable frequency and voltage that is four programmable protocols
Easy to operate – Hands-free operation with foot switch control
Increase the viability of injected embryos
Easy to operate – Hands-free operation with foot switch control
Pre- and post-implantation in embryos of various species – mice, rodents, monkeys, bovine, pigs, zebrafish, etc.
Microinjectionis an active method for
creating transgenic animals. It is easy
to learn and does not require the most expensive equipment. This method
described below is geared towards generating transgenic animals, during the
process of genetic transformation. Transgenic
animals are defined as animals
in which new or altered genes have been experimentally inserted into their
genome by genetic engineering techniques. They are routinely used in the
laboratory as a model in Bio-medical research. Mice are the model of choice,
not only because there is an extensive analysis of its completed genome
sequence, but its genome is similar to the human.
Advantages and disadvantages of transgenic
animals
Advantages:-
1. It has been estimated that transgenic animal can produce in its lifetime $100 to $200 million worth of pharmaceuticals.
2. The isolation and purification of
expressed protein in the conventional method are more difficult than purifying
proteins from an animal’s milk or body fluid.
3. Expression through cell culture or
bacterial culture requires constant monitoring and sampling.
4. It is more cost effective as the
product is efficiently passed through milk with an average yield of 53% and
with 99% purity.
5. Gene requires a certain cellular
mechanism to help for the production of protein.
Disadvantages:-
1. A large number of recipients are required for embryo transfer because of the low transgenesis rate.
2. The generation of transgenic animals is also
expensive, because of long gestation period, litter size and higher maintenance
cost of the recipient animals.
3. The transgenic animal project is extremely
expensive.
Electroporation is the presentation of an electrical current to a living
surface (as the cell membrane or a skin) so asto open pores or channels through that one thing(as a drug or DNA) may
pass.
It is the use of high-voltage electrical shocks to introduce
DNA into cells. It can be used with most cell sorts, yields a high frequency
for each stable transformation and transient organic phenomenon and because it
needs fewer steps, are often easier than alternate techniques. This method
describes procedure for using electroporation in vivo to perform gene therapy
for cancer therapy and DNA vaccination, and outlines modifications for
preparation and transfection of plant protoplasts.
Electroporation could be a helpful methodology for delivering long dsRNA in vivo to immature tick stages whereverinjections and capillary feeding don’t seem to be possible. It is a way that’s conjointly being investigated for dsRNA delivery in tick eggs.
In laboratory, electroporation is finished with Associate in Nursing appliance that makes Associate in Nursingmagnetic attraction field within the cell answer called Associate in Nursing electroporator.
The cell suspension is pipetted into a glass or plastic cuvette that has 2 aluminium electrodes on its sides. Electroporation is employed in several areas of biological science analysis similarly as within the medical field.
Some applications of electroporation embody includes
DNA transfection
or transformation,
direct
transfer of plasmids between cells,
iatrogenic cell
fusion,
trans-dermal
drug delivery,
cancer neoplasm electrochemotherapy
and
cistronmedical aid.
Benefits of electroporation
It is non-viral, non-toxic and can be used on all cell types including mammalian, bacteria, algae, plant and yeast.
It’s reproducible, highly efficient and easy to use.
It is used on cells altogether forms, in vitro or in vivo/ex vivo.
It’s not limited by plasmid size and uptake is immediate and requires no incubation.
In vivo/ex vivo, for “within the living”and includes tissue/whole organ, in ovo, and in utero.
In vitro, for “within glass” and includes suspension cell, tissue slice/whole organ, and adherent cell.
Electroporation Applications
For gene/drug delivery, to study the effects of genes or drugs on cells.
For research, medical, farming and manufacturing process.
Vaccine Development
To stimulate a more robust immune response.
B-cell cloning, for monoclonal antibody production in mammalian cells.
Bacterial Libraries
What can be electroporated?
Bacteria
Fungi/Yeast
Plants
Others, insects, fish, mold, and amphibian Mammalian
A cell is the Structural and Functional unit of Life and in the human body there are different types of cells and none of them are identical in nature. To get clarity on heterogeneity on the cell, various manipulation and comprehensive analysis of cells at single -cell level is being performed. By using traditional techniques such as Petri-dishes or well-plates it was a tedious task to manipulate and analyze single-cells with small size and low concentration of target biomolecules. With the advent of microfluidics, which caters to manipulating and controlling fluids in the range of micro- to pico-liters, a single-cell study has been a boom for almost two decades. Microfluidic single-cell analysis offers benefits of higher throughput, smaller sample volume, automatic sample processing, and lower contamination risk, etc., which has made microfluidics an ideal technology for implementing statically meaningful single-cell research.
Single-cell analysis is, in fact, more troublesome than bulk-cell analysis as far as the sizes of cells and the concentrations of cellular components are concerned. Most of the cells, for example, mammalian and microbes’ cells have sizes at the scale of microns. Therefore, alteration of those cells at the single-cell level becomes a tedious task when traditional biological tools are used such as Petri-dishes and well-plates. Moreover, a large portion of the intracellular, extracellular components are exhibited in little fixations and have a wide scope of concentrations, which require highly sensitive and explicit detection techniques. Many single-cell examination applications require single-cell isolation first, and multi-well plates are commonly used in most biological labs for single-cell isolation, which has low efficiency and requires huge labor. Flow cytometry or laser scanning cytometry, which rapidly screens fluorescently labeled cells in flow, been created and perceived as a brilliant standard for single-cell examination for quite a while. Taking flow cytometry as an example, although they are automatic in nature, capable of multiple detections, and have efficiency in single-cell sorting, they are bulky, mechanically complicated, expensive, and require large sample volumes. Also, they can only be used for analyzing cells at one time-point. Hence, it is impossible to use flow cytometry for continuously tracking cell dynamics. Owing to the capability of manipulating and controlling fluids in the range of micro to pico-liters, microfluidics has been developed as a platform-level and constantly developing innovation for single-cell manipulation and investigation for around two decades.
Microfluidics provides many incomparable advantages over conventional techniques. • Firstly, the microfluidic chip can be flexibly designed to cater to the demands of diverse single-cell manipulation and analysis tasks. For example, single-cell manipulation can be achieved by using either passive or active method, and single-cell analysis can be achieved by implementing either optical or electrochemical method. • Secondly, miniaturized microfluidic systems work can cope up with a very small volume of liquid, which reduces sample loss and decreases dilution leading to sensitive detections. Hence, many microfluidics-based biosensors have been developed. • Thirdly, microfluidics helps in achieving high-throughput parallel manipulation and analysis of the sample, which is beneficial for the statistically meaningful single-cell analysis. • Fourthly various functionalities are effectively coordinated on a similar chip, which leads to automation and this eradicates errors introduced by manual operations.
Specifically, the microfluidic single-cell manipulation can be obtained by using hydrodynamic, electrical, optical, magnetic, and acoustic and micro-robot assisted methods, and microfluidic chips can be combined with different analytical techniques for single-cell analysis ranging from cellular behaviors to secreted proteins. In general, hydrodynamic method caters to high throughput manipulation of cell samples, but it lacks in the accuracy and flexibility. Methods such as electrical and optical methods have high accuracy and great flexibility, but they have deficiencies in throughput. There is no one method that can fulfill high throughput, high-efficiency, accurate single-cell manipulation and analysis tasks simultaneously. Therefore, to achieve the specific requirements of practical applications, multiple methods need to be integrated. Also, existing technologies should be continuously enhanced for better single-cell manipulation and analysis. Lastly, microfluidic chip embedded in subcutaneous, blood vessels and other tissues and organs, for accomplishing precise single-cell manipulation and analysis directly in the human body, is in a growing stage and requires continuous innovation in it.
Microinjection for transgenic animals is a method where the foreign DNA transfers into the animals. A transgenic animal is one whose genome has been altered by the transfer of the genes from another species or breed. Tests of Microinjection for transgenic animals are routinely held in the laboratory and research centers. These animals are most commonly created by the microinjection of DNA into the pronuclei of a fertilized egg. The insertion of DNA isa random process, and there is a high probability that the introduced gene will not insert itself into a site on the DNA, as that will permit its expression. In this process, the manipulated fertilized ovum is been transferred into the oviduct of the foster mother. And that has been induced to act as a recipient by mating with a vasectomized male. Transgenic animals are being explored as a means to produce large quantities of complex human proteins for the treatment of human disease. The therapeutic proteins are currently produced in mammalian cell-based reactors, but this production process is very expensive.
Embryonic stem cell-mediated gene transfer system is the method involves prior insertion of the desired DNA sequence by homologous recombination into an in vitro culture of embryonic stem (ES) cells. These cells are incorporated into an embryo at the blastocyst stage of development. The result is a chimeric animal. ES cell-mediated gene transfer is the method of choice for gene inactivation,so this is the so-called knock-out method. The scientist use retrovirus-mediated gene transfer system to increase the probability of expression.
The gene transfer is a process mediated by the means of a carrier or a vector, generally a virus or a plasmid. Whilemost of these transgenic animals arespecifically used to identify the functions of some specific factors in the homeostatic systems through the overor under-expression of a modified genein the mammalian developmental genetics and also in the molecular biology.
Transgenic Zebra fish
At the level of the whole animal, like the transgenic mice or zebra fish, the analysis of such regulation of the gene expression makes use of the evaluation of a specific genetic change. As for an example in the pharmaceutical industry, the targeted production of pharmaceutical proteins and drug production, product efficacy testing in biotechnology in one such usage area. Let us discuss some of the other application areas, such as the genetically engineered hormones that helps to increase milk yield, meat production; genetic engineering of livestock and in aquaculture affecting modification of animal physiology. The cloning procedures to reproduce specific blood lines and developing animals specially created for use in xenografting is another important usage area.
These animals are generally established by traditional zygote-injection-based technology. Transgenic rat models are established only by the former technology because of a lack of rat cell lines.
Transgenic Rat
Advantages and disadvantages of transgenic animals
Advantages:-
1. It has been estimated that
transgenic animal can produce in its lifetime $100 to $200 million worth of
pharmaceuticals.
2.
The isolation and purification of expressed protein in conventional method is
more difficult than purifying proteins from an animal’s milk or body fluid.
3.
Expression through cell culture or bacterial culture requires constant
monitoring and sampling.
4.
It is more cost effective as the product is efficiently passed through milk
with an average yield of 53% and with 99% purity.
5. Gene requires certain cellular mechanism to help for the production of protein. While such proteins in animals are used for transgenic purpose that naturally carries the mechanism, that are needed to produce some complex protein. These mechanism is just absent in the cell culture.
Disadvantages:-
1.
Large number of recipients is required for embryo transfer because of low
transgenesis rate.
2. Generation of transgenic animals are also expensive, because of long gestation period, litter size and higher maintenance cost of the recipient animals.
3. Transgenic animal project is extremely expensive.
Pinpoint Cell Penetrator for Targeted Microinjection 