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Can the activity of a specific gene in a cell artficially be increased?

Can the activity of a specific gene in a cell artficially be increased?



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While there are many gene regulation mechanisms from the cell itself, I was wondering whether it is possible to increase the gene activity in a living cell permanently (so that the protein that it codes for is produced in higher amounts) using methods of biotechnology (i.e. no external factors)? Are there any studies or experiments regarding this topic?


There are technologies available for inducing higher (or lower) gene expression in cultured mammalian/human cells as well as in bacteria. The state of the art is CRISPR-mediated gene activation/repression, where you fuse a transcription activation domain to a Cas gene in a CRISPR-Cas system (usually Cas9), and provide a guide RNA to target genes of interest. This Cas gene is made "nuclease-dead" so that it can only target but not cut or degrade DNA (as opposed to natural CRISPR-Cas systems that do both), and serves to bring the transcription activation domain to your gene of interest, which leads to an increase in transcription of that gene.

Check out this paper for an earlier example of such a system in cultured cell lines. The field has advanced quite a bit since then, and people have successfully activated genes in mouse lines


If you are after large amounts of a particular protein, search "recombinant protein production". It is a huge field.


A sequence is executed with transcriptase. To double amount of signaling or some protein production try doubling the sequence occurrence.


It can be done. I've found this article which describes how cou can use a modified CRISPR-Cas9 mechanism to either repress or activate transcription.


Design of Vectors for Optimizing Transgene Expression

B Gene Inactivation

Gene knockout is a potent and irreversible means to inactivate a gene. The Cre-LoxP system is one possibility (see Section 17.V.A ). The knockout can be achieved using conventional homologous recombination or with engineered endonucleases (see Section 17.IV ). A gene knockout may also be obtained using the NHEJ after a double break of DNA at the chosen site using engineered endonucleases or the RGEN system (see Section 17.IV ). The advantages of this approach are its high efficiency and the fact that the inactivation may be achieved in one-cell embryos.


Molecular pathogenesis of oral squamous cell carcinoma: implications for therapy

The development of oral squamous cell carcinoma (OSCC) is a multistep process requiring the accumulation of multiple genetic alterations, influenced by a patient's genetic predisposition as well as by environmental influences, including tobacco, alcohol, chronic inflammation, and viral infection. Tumorigenic genetic alterations consist of two major types: tumor suppressor genes, which promote tumor development when inactivated and oncogenes, which promote tumor development when activated. Tumor suppressor genes can be inactivated through genetic events such as mutation, loss of heterozygosity, or deletion, or by epigenetic modifications such as DNA methylation or chromatin remodeling. Oncogenes can be activated through overexpression due to gene amplification, increased transcription, or changes in structure due to mutations that lead to increased transforming activity. This review focuses on the molecular mechanisms of oral carcinogenesis and the use of biologic therapy to specifically target molecules altered in OSCC. The rapid progress that has been made in our understanding of the molecular alterations contributing to the development of OSCC is leading to improvements in the early diagnosis of tumors and the refinement of biologic treatments individualized to the specific characteristics of a patient's tumor.


A new mechanism behind continuous stem cell activity in plants

Figure 1: Diagrams of the vascular genetic expression network (Purple= Xylem cells, Green= Phloem Cells, Blue= Vascular stem cells). A. Vascular development during the plant’s secondary growth. B. The vascular cell induction culture system ‘VISUAL’. C. The constructed vascular gene expression network. Each dot indicates a gene and the lines show strong mutual relationships between them. Credit: Kobe University

An inter-university research group has succeeded in constructing the gene expression network behind the vascular development process in plants. They achieved this by performing bioinformatics analysis using the 'VISUAL' tissue culture platform, which generates vascular stem cells from leaf cells. In this network, they also discovered a new BES/BZR transcription factor, BEH3, which regulates vascular stem cells. In addition, they illuminated a novel vascular cell maintenance system whereby BEH3 competes with other transcription factors from the same BES/BZR family in order to stabilize vascular stem cell multiplication and differentiation.

The joint research group consisted of scientific researcher Furuya Tomoyuki and Associate Professor Kondo Yuki et al. (of Kobe University's Graduate School of Science), Kyushu University's Professor Satake Akiko, Specially Appointed Professor Tanokura Masaru and Specially Appointed Associate Professor Miyakawa Takuya (of the University of Tokyo's Graduate School of Agricultural and Life Sciences), and Associate Professor Yamori Wataru (of the University of Tokyo's Institute for Sustainable Agro-ecosystem Services).

The researchers hope to identify more regulatory factors for stem cells, which will contribute towards our understanding of the molecular basis behind continuous stem cell activity in plants.

These research results were published in the American plant sciences journal The Plant Cell on June 1, 2021.

  • The researchers extracted a total of 394 genes specific to vascular stem cells from extensive gene expression datasets. Among these, they discovered BEH3, a novel stem cell regulatory factor belonging to the BES/BZR family of transcription factors.
  • They discovered that unlike the other BES/BZR transcription factors, BEH3 has almost no functional domains and competitively inhibits the activity of these other factors.
  • The research group showed that this competitive relationship between BES/BZR transcription factors stabilizes the multiplication and differentiation of vascular stem cells, illuminating the regulatory system that maintains vascular stem cells' continuous activity.
Figure 2: Model diagram showing the competitive relationship between BEH3 and other BES/BZR transcription factors. BEH3 and the other BES/BZR transcription factors (here represented by BES1) compete with each other to bind to the DNA motif BRRE, regulating downstream gene expression. Credit: Kobe University

Plants take form by self-replicating their stem cells and differentiating these stem cells so that they have specialized functions for constructing parts of the plant, such as its organs and tissues. Unlike animals, plants continue to regenerate and grow by producing stem cells throughout their life. For example, trees such as cryptomeria can have long lifespans (the Jomon Cedar Tree on Japan's Yakushima Island is at least 2000 years old), and each year they promote secondary growth which results in another tree ring around their trunks. This secondary growth is occurs inside a region of meristem tissue called the cambium layer where vascular stem cells multiply and differentiate into xylem cells and phloem cells, enabling the trunk to grow wider. In other words, plant must continuously produce vascular stem cells throughout their lives in order to keep growing, and it is vital for them to maintain the balance between stem cell multiplication and differentiation.

In recent years, studies using the model plant Arabidopsis thaliana have been conducted into how the multiplication and differentiation of stem cells are regulated from the perspectives of genetics, life sciences and informatics research. However, the mechanism by which plants regulate and maintain the appropriate balance of stem cells has yet to be understood.

Research Methodology and Findings

In order to analyze the process by which vascular stem cells differentiate into xylem cells and phloem cells (Figure 1), Associate Professor Kondo et al."s research group developed the tissue culture system 'VISUAL' to artificially generate stem cells from leaf cells. VISUAL has many benefits that make it suitable for research on vascular stem cells, for example, it is easy to genetically analyze plants that have a particular gene function removed (i.e. mutants) and it is also possible to observe the temporal progression of vascular stem cell differentiation. In this study, the researchers collected data on multiple mutants and carried out large-scale analyses of gene expression at various time points. They conducted gene co-expression network analysis on similarities in the expression patterns to evaluate the relationship between different genes. From this analysis, they succeeded in identifying the distinctive groups of genes in xylem cells, phloem cells and vascular stem cells (Figure 1). Using VISUAL, this research group previously revealed that the BES/BZR transcription factors BES1 and BZR1 play an important role in vascular stem cell differentiation. This time, they identified another BES/BZR transcription factor, BEH3, in the vascular stem cell gene group through network analysis, and also examined its vascular stem cell suppressing function.

Figure 3: A model of vascular stem cell regulation based on this research. Competition within the BES/BZR transcription factor family robustly regulates the balance between vascular stem cell multiplication and differentiation, contributing towards the maintenance of continuous stem cell activity. Credit: Kobe University

Next, the researchers investigated vascular formation using mutants with BEH3's function removed. They found that the mutants had large variations in vascular size compared to the wild type (non-mutant plant) and concluded that BEH3 stabilizes vascular stem cells. The research group had previously found that strengthening the function of BES1 (which promotes vascular cell differentiation) caused the number of vascular cells to decrease, however they found that when they strengthened the function of BEH3 opposite occurred and the number of vascular stem cells increased. Upon researching this further, the research group discovered that even though BEH3 could bind to the same DNA motif as the other BES/BZR transcription factors, BEH3's ability to regulate the expression of downstream genes was significantly weaker. These results showed that BEH3 hinders the activity of other BES/BZR transcription factors (Figure 2), and the researchers inferred from this relationship that BEH3's function in vascular stem cells is opposed to that of the factors in the same family, including BES1. A mathematical model was used to verify and simulate this competitive relationship between BEH3 and the other BES/BZR transcription factors, and the results indicated that the presence of BEH3 in vascular stem cells contributes towards stabilizing vascular size (Figure 3).

There are thought to be many important gene candidates in this research group's vascular stem cell gene expression network that will contribute towards understanding of vascular development and functions. It is hoped that the valuable information obtained through this study will accelerate vascular research. In addition, further illuminating the relationships between BEH3 and other BES/BZR transcription factors and their respective differences will deepen our understanding of the mechanism by which plants maintain the balance between stem cell multiplication and differentiation.

In the future, this knowledge could contribute towards biomass production techniques, and other areas that require large-scale stable plant growth.


Relationship of cell growth to the regulation of tissue-specific gene expression during osteoblast differentiation

The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation can be examined in primary diploid cultures of fetal calvarial-derived osteoblasts by the combination of molecular, biochemical, histochemical, and ultrastructural approaches. Modifications in gene expression define a developmental sequence that has 1) three principal periods: proliferation, extracellular matrix maturation, and mineralization and 2) two restriction points to which the cells can progress but cannot pass without further signals. The first restriction point is when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle and cell growth regulated genes, produce a fibronectin/type I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which an enhanced expression of alkaline phosphatase occurs immediately after the proliferative period, and later an increased expression of osteocalcin and osteopontin at the onset of mineralization 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited and 3) enhanced levels of expression of the osteoblast markers when collagen deposition is promoted, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and development of the osteoblast phenotype. The loss of stringent growth control in transformed osteoblasts and in osteosarcoma cells is accompanied by a deregulation of the tightly coupled relationship between proliferation and progressive expression of genes associated with bone cell differentiation.— S tein , G. S. L ian , J. B. O wen , T. A. Relationship of cell growth to the regulation of tissue-specific gene expression during osteoblast differentiation. FASEB J. 4: 3111-3123 1990.


MATERIALS AND METHODS

Data processing and analysis

For each expression compendium (HBI [GEO Accession No. <"type":"entrez-geo","attrs":<"text":"GSE7307","term_id":"7307">> GSE7307], expO [GEO Accession No. <"type":"entrez-geo","attrs":<"text":"GSE2109","term_id":"2109">> GSE2109], and CCLE [www.broadinstitute.org/ccle]), Affymetrix Human Genome U133 Plus 2.0 arrays were normalized with robust multiarray average (RMA) using the affy package from Bioconductor (www.bioconductor.org/packages/release/bioc/html/affy.html) and Entrez Gene (www.ncbi.nlm.nih.gov/gene) ID custom probeset definitions as defined previously ( Dai etਊl., 2005 ). The RMA values of selected genes were subsequently median centered and hierarchically clustered (based on uncentered correlation and average linkage) using Cluster 3.0 ( de Hoon etਊl., 2004 ) and visualized with Java TreeView ( Saldanha, 2004 ). Principal component analysis plots gave similar results to the hierarchical clustering (unpublished data).

For the cell cycle study ( Grant etਊl., 2013 ), processed Agilent Whole Human Genome Oligonucleotide array ratios (sample/reference) were downloaded from Supplemental Table S1. The profiles of selected genes were sorted by arctan2 values, as provided in the table.

For TCGA breast invasive carcinoma data, processed Agilent expression data (level 3) for matching normal and tumor pairs (sample IDs are given in Table 1 ) were downloaded from the TCGA data portal (https://tcga-data.nci.nih.gov). For each cancer sample, log2 ratios of selected genes were calculated relative to the matching normal sample, and the heatmap was created as described.

TABLE 1:

TCGA tumor samplesTCGA normal samples
TCGA-A7-A13E-01A-11R-A12P-07TCGA-A7-A13E-11A-61R-A12P-07
TCGA-A7-A13F-01A-11R-A12P-07TCGA-A7-A13F-11A-42R-A12P-07
TCGA-BH-A0AU-01A-11R-A12P-07TCGA-BH-A0AU-11A-11R-A12P-07
TCGA-BH-A0B5-01A-11R-A12P-07TCGA-BH-A0B5-11A-23R-A12P-07
TCGA-BH-A0BS-01A-11R-A12P-07TCGA-BH-A0BS-11A-11R-A12P-07
TCGA-BH-A0BZ-01A-31R-A12P-07TCGA-BH-A0BZ-11A-61R-A12P-07
TCGA-BH-A0C3-01A-21R-A12P-07TCGA-BH-A0C3-11A-23R-A12P-07
TCGA-BH-A0DD-01A-31R-A12P-07TCGA-BH-A0DD-11A-23R-A12P-07
TCGA-BH-A0DT-01A-21R-A12D-07TCGA-BH-A0DT-11A-12R-A12D-07
TCGA-BH-A0HA-01A-11R-A12P-07TCGA-BH-A0HA-11A-31R-A12P-07
TCGA-BH-A18J-01A-11R-A12D-07TCGA-BH-A18J-11A-31R-A12D-07
TCGA-BH-A18K-01A-11R-A12D-07TCGA-BH-A18K-11A-13R-A12D-07
TCGA-BH-A18L-01A-32R-A12D-07TCGA-BH-A18L-11A-42R-A12D-07
TCGA-BH-A18M-01A-11R-A12D-07TCGA-BH-A18M-11A-33R-A12D-07
TCGA-BH-A18N-01A-11R-A12D-07TCGA-BH-A18N-11A-43R-A12D-07
TCGA-BH-A18P-01A-11R-A12D-07TCGA-BH-A18P-11A-43R-A12D-07
TCGA-BH-A18Q-01A-12R-A12D-07TCGA-BH-A18Q-11A-34R-A12D-07
TCGA-BH-A18R-01A-11R-A12D-07TCGA-BH-A18R-11A-42R-A12D-07
TCGA-BH-A18S-01A-11R-A12D-07TCGA-BH-A18S-11A-43R-A12D-07
TCGA-BH-A18U-01A-21R-A12D-07TCGA-BH-A18U-11A-23R-A12D-07
TCGA-BH-A18V-01A-11R-A12D-07TCGA-BH-A18V-11A-52R-A12D-07
TCGA-BH-A1EO-01A-11R-A137-07TCGA-BH-A1EO-11A-31R-A137-07
TCGA-BH-A1EU-01A-11R-A137-07TCGA-BH-A1EU-11A-23R-A137-07
TCGA-E2-A153-01A-12R-A12D-07TCGA-E2-A153-11A-31R-A12D-07
TCGA-E2-A158-01A-11R-A12D-07TCGA-E2-A158-11A-22R-A12D-07
TCGA-E2-A15I-01A-21R-A137-07TCGA-E2-A15I-11A-32R-A137-07
TCGA-E2-A15M-01A-11R-A12D-07TCGA-E2-A15M-11A-22R-A12D-07
TCGA-E2-A1BC-01A-11R-A12P-07TCGA-E2-A1BC-11A-32R-A12P-07

A subset of the expO tumor samples was also compared with normal counterparts in the HBI compendium. For each of the common HBI tissues (kidney, colon, liver, breast, lung, ovary/uterus, and prostate), the median expression level of each gene in that tissue was calculated, and each expO tumor sample was compared with the set of corresponding normal medians. The heatmap was generated as described.

Pearson correlations were calculated in R by comparing the expression (log2 ratios) of each gene against every other gene in the data set. The correlation values were then clustered in Cluster3 and visualized in Java TreeView, as described. Correlations were summarized by the mean via Fisher transformation.


Contents

DCas9 Edit

Cas9 Endonuclease Dead, also known as dead Cas9 or dCas9, is a mutant form of Cas9 whose endonuclease activity is removed through point mutations in its endonuclease domains. Similar to its unmutated form, dCas9 is used in CRISPR systems along with gRNAs to target specific genes or nucleotides complementary to the gRNA with PAM sequences that allow Cas9 to bind. Cas9 ordinarily has 2 endonuclease domains called the RuvC and HNH domains. The point mutations D10A and H840A change 2 important residues for endonuclease activity that ultimately results in its deactivation. Although dCas9 lacks endonuclease activity, it is still capable of binding to its guide RNA and the DNA strand that is being targeted because such binding is managed by other domains. This alone is often enough to attenuate if not outright block transcription of the targeted gene if the gRNA positions dCas9 in a way that prevents transcriptional factors and RNA polymerase from accessing the DNA. However, this ability to bind DNA can also be exploited for activation since dCas9 has modifiable regions, typically the N and C terminus of the protein, that can be used to attach transcriptional activators. [2]

Guide RNA Edit

A small guide RNA (sgRNA), or gRNA is an RNA with around 20 nucleotides used to direct Cas9 or dCas9 to their targets. gRNAs contain two major regions of importance for CRISPR systems: the scaffold and spacer regions. The spacer region has nucleotides that are complementary to those found on the target genes, often in the promoter region. The scaffold region is responsible for formation of a complex with (d)Cas9. Together, they bind (d)Cas9 and direct it to the gene(s) of interest. Since the spacer region of a gRNA can be modified for any potential sequence, they give CRISPR systems much more flexibility as any genes and nucleotides with a sequence complementary to the spacer region can become possible targets. [2]

Transcriptional activators Edit

Transcriptional Activators are protein domains or whole proteins linked to dCas9 or sgRNAs that assist in the recruitment of important co-factors as well as RNA Polymerase for transcription of the gene(s) targeted by the system. In order for a protein to be made from the gene that encodes it, RNA polymerase must make RNA from the DNA template of the gene during a process called transcription. Transcriptional activators have a DNA binding domain and a domain for activation of transcription. The activation domain can recruit general transcription factors or RNA polymerase to the gene sequence. Activation domains can also function by facilitating transcription by stalled RNA polymerases, and in eukaryotes can act to move nucleosomes on the DNA or modify histones to increase gene expression. [3] These activators can be introduced into the system through attachment to dCas9 or to the sgRNA. Some researchers have noted that the extent of transcriptional upregulation can be modulated by using multiple sites for activator attachment in one experiment and by using different variations and combinations of activators at once in a given experiment or sample. [4] [5] [6]

Expression system Edit

An expression system is required for the introduction of the gRNAs and (d)Cas9 proteins into the cells of interest. Typically employed options include but are not limited to plasmids and viral vectors such as adeno-associated virus (AAV) vector or lentivirus vector.

VP64-p65-Rta Edit

The VP64-p65-Rta, or VPR, dCas9 activator was created by modifying an existing dCas9 activator, in which a Vp64 transcriptional activator is joined to the C terminus of dCas9. In the dCas9-VPR protein, the transcription factors p65 and Rta are added to the C terminus of dCas9-Vp64. Therefore, all three transcription factors are targeted to the same gene. The use of three transcription factors, as opposed to solely Vp64, results in increased expression of targeted genes. When different genes were targeted by dCas9, they all showed significantly greater expression with dCas9-VPR than with dCas9-VP64. It has also been demonstrated that dCas9-VPR can be used to increase expression of multiple genes within the same cell by putting multiple sgRNAs into the same cell. [7] dCas9-VPR has been used to activate the neurogenin 2 (link) and neurogenic differentiation 1 (link) genes, resulting in differentiation of induced pluripotent stem cells into induced neurons. [7] A study comparing dCas9 activators found that the VPR, SAM, and Suntag activators worked best with dCas9 to increase gene expression in a variety of fruit fly, mouse, and human cell types. [8]

Synergistic activation mediator Edit

To overcome the limitation of the dCas9-VP64 gene activation system, the dCas9-SAM system was developed to incorporate multiple transcriptional factors. Utilizing MS2, p65, and HSF1 proteins, dCas9-SAM system recruits various transcriptional factors working synergistically to activate the gene of interest.

In order to assemble different transcriptional activators, the dCas9-SAM system uses a modified single guide RNA (sgRNA) that has binding sites for the MS2 protein. Hairpin aptamers are attached to the tetra loop and the stem loop 2 of the sgRNA to become binding sites for dimerized MS2 bacteriophage coat proteins. As the hairpins are exposed outside of the dCas9-sgRNA complex, other transcriptional factors can bind to the MS2 protein without disrupting the dCas9-sgRNA complex. Thus, the MS2 protein is engineered to include p65 and HSF1 proteins. The MS2-p65-HSF1 fusion protein interacts with the dCas9-VP64 to recruit more transcriptional factors onto the promoter of the target genes.

Employing the dCas-SAM system, Zhang et al. (2015) successfully reactivated the latent HIV gene to over-express viral proteins from the HIV host cells. [9] They were able to over-express viral proteins substantially to trigger apoptosis of HIV-1 latent cells due to the toxicity of viral proteins. In another dCas-SAM system experiment, Konermann et al. (2015) found genes in melanoma cells that give resistance to a BRAF inhibitor through activating candidate genes via dCas system. [6] Thus, the dCas9-SAM system can further be employed to activate latent genes, develop gene therapies, and discover new genes.

SunTag Edit

The SunTag activator system uses the dCas9 protein, which is modified to be linked with the SunTag. The SunTag is a repeating polypeptide array that can recruit multiple copies of antibodies. Through attaching transcriptional factors on the antibodies, the SunTag dCas9 activating complex amplifies its recruitment of transcriptional factors. In order to guide the dCas9 protein to its target gene, the dCas9 SunTag system uses sgRNA.

Tanenbaum et al.(2014) are credited for creating the dCas9 SunTag system. For the antibodies, they employed GCN4 antibodies which was bound to transcriptional factor VP64. In order to transport the antibodies to the nuclei of the cells, they attached NLS tag. To confirm the nuclear localization of the antibodies, sfGFP was used for visualization purpose. Therefore, the GCN4-sfGFP-NLS-VP64 protein was developed to be interact with dCas SunTag system. The antibodies successfully bound to SunTag polypeptides and activated target CXCR4 gene in K562 cell lines. [10] Comparing with the dCas9-VP64 activation complex, they were able to increase the CXCR4 gene expression 5-25 times greater in K562 cell lines. Not only was there a greater CXCR4 protein overexpression but also CXCR4 proteins were active to further travel on the transwell migration assay. Thus, the dCas9-SunTag system can be used to activate genes that are present latently such as virus genes.

The dCas9 activation system allows a desired gene or multiple genes in the same cell to be expressed. It is possible to study genes involved in a certain process using a genome wide screen that involves activating expression of genes. Examining which sgRNAs yield a phenotype suggests which genes are involved in a specific pathway. The dCas9 activation system can be used to control exactly which cells are activated and at what time activation occurs. dCas9 constructs have been made that turn on a dCas9-activator fusion protein in the presence of light or chemicals. Cells can also be reprogrammed or differentiated from one cell type into another by increasing the expression of certain genes important for the formation or maintenance of a cell type. [11]

Greater control over gene expression Edit

One research group used a system in which dCas9 was fused to a particular domain, C1B1. When blue light is shined on the cell, the Cry2 domain binds to C1B1. The Cry2 domain is fused to a transcriptional activator, so blue light targets the activator to the spot where dCas9 is bound. The use of light allows a great deal of control over when the targeted gene is activated. Removing the light from the cell results in only dCas9 remaining at the target gene, so expression is not increased. In this way, the system is reversible. [12] A similar system was developed using chemical control. In this system, dCas9 recruits an MS2 fusion protein that contains the domain FKBP. In the presence of the chemical RAP, an FRB domain fused to a chromatin modifying complex binds to FKBP. Whenever RAP is added to the cells, a specific chromatin modifier complex can be targeted to the gene. That allows scientists to examine how specific chromatin modifications affect the expression of a gene. [13] The dCAs9-VPR system is used as an activator by targeting it to the promoter of a gene upstream of the coding region. A study used various sgRNAs to target different portions of the gene, finding that the dCas9-VPR activator can act as an activator or a repressor, depending on the location it binds. In a cell, sgRNAs targeting the promoter could allow dCas9-VPR to increase expression, while sgRNAs targeting the coding region of the gene result in dCas9-VPR decreasing expression. [14]

Genome wide activation Edit

The versatility of sgRNAs allows dCas9 activators to increase the expression of any gene within an organism's genome. That could be used to increase expression of a protein coding gene or a transcribed RNA. A paper demonstrated that genome wide activation could be used to determine which proteins are involved in mediated resistance to a specific drug. [6] Another paper used genome wide activation of long, noncoding RNAs and observed that increasing the expression of certain long noncoding RNAs conferred resistance to the drug vemurafenib. [15] In both cases, the cells that survive the drug could be studied to determine which sgRNAs they contain. That allows researchers to determine which gene was activated in each surviving cell, which suggests which genes are important for resistance to that drug.

Use in organisms Edit

A dCas9 fusion with VP64, p65, and HSF1 (heat shock factor 1) allowed researchers to target genes in Arabidopsis thaliana and increase transcription to a similar level as when the gene itself is inserted into the plant's genome. For one of the two genes tested, the dCas9 activator changes the number and size of leaves and made the plants better able to handle drought. The authors conclude that the dCas9 activator can create phenotypes in plants that are similar to those observed when a transgene is inserted for overexpression. [16] Researchers have used multiple guide RNAs to target dCas9 activation system to multiple genes in a specific mouse strain in which dCas9 can be turned on in specific cell lines using the Cre recombinase system. Scientists used the targeting and increased expression of several genes to examine the processes involved in regeneration and carcinomas of the liver. [17]


Genetic Engineering for Plant Transgenesis

Surender Khatodia , S.M. Paul Khurana , in Omics Technologies and Bio-Engineering , 2018

Abstract

Genetic engineering of plants to make a production system for recombinant proteins to produce medicinal and industrial compounds of human value is called Plant Molecular Farming. Using plants as bioreactor for recombinant protein production has several advantages, including the low cost of establishment, and the scalability of plants and products generally regarded as safe. Plant-made pharmaceuticals (PMPs) are already being produced commercially for some diseases like Gaucher’s disease, and many plant-made vaccines are under clinical trials. Two major classes of pharmaceuticals are in development using genetic engineering of plants—Plantibodies and Edible vaccines. This chapter highlights the procedure of using plants as bioreactor for recombinant protein production through plants, increasing recombinant protein accumulation, commercial status of PMPs and chloroplast genome engineering.


Discussion

A cell-based reporter system was established for demonstrating hammerhead and glmS ribozyme activity via the assessment of reporter protein production. The established E. coli cells contained two foreign genes encoding EGFP-hDHFR reporter protein and RNaseJ1 ribonuclease enzyme. Initially, expression of the proteins was tested in single plasmid transformant or co-transformant cells. Highly variable growth and reporter production were observed in these transformants (Fig. 2b and Table S3) that reduced power to detect ribozyme activity. Plasmids impose a metabolic burden on transformed E. coli owing to the expression of plasmid encoded genes and replication of plasmid DNA [29]. The overproduction of RNaseJ1 inhibits growth of the pRJ1 plasmid transformant (Fig. S1). RNaseJ1 functions as a 5′-3′ exonuclease enzyme which plays a pivotal role in B. subtilis RNA metabolism. Although RNaseJ1 is not present in E. coli, it has some functional overlap with E. coli RNaseE [30]. The overexpression of RNaseJ1 in E. coli transformants carrying pRJ1 plasmid thus might have an impact on general RNA metabolism, affecting cell homeostasis and growth.

The degree of the metabolic load in plasmid transformants is also dependent on plasmid copy number and size [29]. Poor growth of co-transformants over-expressing RNaseJ1 (Fig. 2b) could also be exacerbated by the extra burden of carrying two plasmid types lacking segregation control. The reporter gene-expressing plasmids contain a pUC origin of replication, whereas the RNaseJ1-expressing plasmid contains a p15A origin of replication (Fig. 1). Plasmid copy numbers of these two replication systems are comparable [31]. However, the transformant population is heterogeneous owing to the unequal distribution of cellular components in cell division [32, 33], which is manifest as a bimodal distribution of plasmid copy number [31]. To mitigate metabolic burden, the heterologous genes were stably integrated via homologous recombination into the E. coli chromosome. Previous studies have demonstrated that genome integration of foreign genes is superior to plasmid-based expression, in which the heterologous protein is expressed more stably and with less impact on growth [34,35,36]. In concordance with previous studies showing the benefit of gene integration, the growth of iRJ1 with integrated RNaseJ1 gene transformed with different reporter plasmids was more consistent compared with plasmid co-transformants, with only minor retardation in the induced condition (Fig. 4b). However, induction of RNaseJ1 expression in iRJ1 transformants had a small and unspecific effect on plasmid-expressed reporter signal such that it was not possible to demonstrate ribozyme activity (Fig. 4c). We do not know why ribozyme activity could not be demonstrated with plasmid-based reporters, but the combined metabolic burden of carrying reporter plasmids and expressing heterologous proteins may create a cellular condition in which RNaseJ1 is less active and/or mRNA is globally more stable.

In contrast to iRJ1 plasmid transformants, specific effects of upstream active ribozyme sequences on reporter activity were demonstrated in double integrants. However, the mean level of reporter protein for the +ara condition was less than two-fold different from the corresponding −ara condition (Fig. 5b). This suggests that the degradation of ribozyme-cleaved reporter RNA by RNaseJ1 has a modest effect on the level of reporter protein under the assay conditions used. Although degradation of ribozyme-cleaved RNA by RNaseJ1 is rapid in E. coli [23], the reporter protein used in our assays may be stable owing to the EGFP moiety that has a half-life of more than 24 h in E. coli [37]. The reporter protein signal could thus persist even after RNaseJ1-mediated mRNA degradation. Reporter protein stability can be reduced by fusing with a degron, or degradation tag for in vivo proteolysis. For example, C-terminal fusion of the ssrA peptide degron can direct fusion proteins for degradation by the endogenous ClpXP and ClpAP proteases, leading to rapid protein degradation in E. coli [38]. The reporter protein with a shorter half-life will be beneficial for widening the dynamic range of the reporter assay. In addition, the ribozyme activity can be monitored in real time along with bacterial growth. As an alternative to protein reporters, turn-on fluorescent RNA reporters could be used [39]. RNA reporters expressed on the same RNA molecule as the ribozyme could give a better time-resolved signal of ribozyme activity, although extensive empirical testing may be required to establish a general ribozyme reporter system. Turn-on fluorescent RNA reporters are generally less bright and thus more difficult to quantify than fluorescent proteins [39], although their performance can be improved by flanking scaffolds, which act by stabilizing the in vivo folding of the RNA reporter [40]. However, scaffolded RNA reporters are more resistant to ribonucleases [40], which may interfere with the detection of ribozyme activity as RNaseJ1-mediated loss of reporter after ribozyme cleavage.

Using the double-integrant reporter system, we demonstrated the activities of the constitutively active (RzI) and cofactor-dependent (glmS) ribozyme. The latter was surprising as no exogenous cofactor was added in the assay, suggesting that the intracellular level of the glmS cofactor, GlcN6P, is sufficiently high for in vivo ribozyme activity. The level of GlcN6P in E. coli varies from 0.062 to 9 mM, depending on the available carbon source [41]. This concentration range is sufficient to activate the glmS ribozyme in vitro [13]. In other cell types such as Saccharomyces cerevisiae yeast [42] and Plasmodium falciparum malaria parasite [43], the glmS ribozyme is inactive unless the intracellular GlcN6P level is increased by treatment with exogenous sugar that can be converted to GlcN6P. The glmS ribozyme cleaves more slowly in vitro than the hammerhead ribozyme at physiological concentrations (≈ 1 mM) of magnesium (glmS rate constant (Kobs) < 1 min − 1 , [13, 44] hammerhead Kobs > 1.2 min − 1 [7]), which could partly explain why the reduction of reporter in the induced condition is less for iglmS_iRJ1 than iRzI_iRJ1 (Fig. 5b). It would be interesting to test other classes of ribozymes for further comparison in our reporter system, including the rapidly cleaving twister ribozyme (twister Kobs ≈ 1000 min − 1 , [10]).


4 DISCUSSION

Planarians are extremely resilient to the perturbation of cell homeostasis processes, including traumatic events such as extensive tissue damage. Their sturdiness resides in the adaptive response of a plastic body where homeostatic processes are continuously adjusted by cross-talk between stem cells and differentiated body parts, which have been suggested to represent a global stem cell niche. 31 For these reasons, unbalancing regenerative and homeostatic performances of planarians is challenging. Accordingly, artificially altered gravity did not permanently perturb planarian regeneration even in extreme conditions. Indeed, animals were able to complete a functional regeneration, although we observed a reduced regenerative capability in head fragments following exposure to simulated hypergravity. Higher susceptibility of head fragments – with respect to tail fragments – to factors perturbing stem cell activity has been commonly demonstrated for these organisms. 58 This phenomenon is probably due to the lower number of stem cells spread in this part of the body, so that minimal variations in the number of neoblasts induce a more readily observable phenotype. In general terms, a reduction in the regeneration ability might owe to reduced cell proliferation and/or an increased cell death. These two biological phenomena are indeed finely regulated during planarian regeneration, both showing two waves of activity, that is, one early after cut and the other later. While apoptosis and proliferation share this biphasic pattern, their distribution in the body is opposite. For apoptosis, the early peak occurs near the wound surface, and the second one is dispersed throughout the body 59 the exact contrary occurs for proliferation, being mitotic cells accumulated under the growing blastema only in the second proliferation burst. 60 A maximal impact upon blastema growth is therefore expected in case of an increase in the first peak of apoptosis and of a decrease in the second peak of proliferation. Slight changes in the number of apoptotic or mitotic cells spread far from the regeneration site are unlikely to directly perturb blastema growth.

In all altered gravity conditions tested in this study, we monitored a reduction of the proliferative activity in the second peak, which is consistent with a reduced blastema growth. On the contrary, we monitored an increase in apoptotic cells under the wound surface in early regeneration only in artificial hypergravity conditions. In the RPM, the number of apoptotic cells localized below the wound is slightly reduced. This difference might be at the basis for the different effect upon blastema growth observed in microgravity versus hypergravity simulations. Indeed, in this latter case the combination of two adverse effects, that is a reduced proliferation at the blastema site and an increased apoptosis under the wound surface, might concur to the slight reduction of the blastema growth rate. We currently do not know which cells underwent apoptosis, but, as at the same time (6 h) at which we observed an increase in apoptosis, we also monitored a number of mitosis comparable to controls so, it is possible to hypothesize that this process preferentially affects post-mitotic cells. Accordingly, it has been demonstrated that the two regenerative apoptotic waves predominantly, if not exclusively, occurred in differentiated cells. 59 We can thus imagine that a first early consequence of hypergravity is the induction of cell-death processes: this is also in line with what we observed in intact animals, where an increment in TUNEL-positive cells was monitored after 24 h of 10 g exposure. Altered gravity is also known to influence cell determination and differentiation in different types of cells ( 22, 61-64 ), and it also has an effect on cell differentiation in planarians, a process that can be easily followed by studying differentiation towards the epidermal lineage, for which early and late molecular markers are available. 50 Obtained data show again an opposite effect of hypergravity and microgravity experiments. Indeed, a significant increase of NB-21.11.e-positive cells (marker for enhanced differentiation) was detectable after simulated hypergravity exposure, while its significant reduction was produced by artificial microgravity exposure indicating a possible demonstration of the gravity continuum paradigm. 65

Pre-treatment with CONPs rescued the negative impact of simulated altered gravity on the blastema growth, cell proliferation, apoptosis, and cell differentiation. We can assume that protective effect is due to internalized nanoparticles, as we extensively washed planarian before exposition to altered gravity conditions. Owing to concomitance on CONP crystalline surface of both Ce 4+ and Ce 3+ states, 40 CONPs show self-regenerating antioxidant properties and ROS-scavenger functions, resulting into anti-inflammatory activity. 66 Hence, CONPs have had several biomedical applications, and showed a protective role in disorders characterized by ROS overproduction. 67-69 Since several literature sources report on overproduction of ROS induced by altered gravity, 70-73 we hypothesize that oxidative unbalance might be the principal cellular stress at which planarian cells are exposed following altered gravity, despite additional concurrent causes cannot be ruled out. Thus, we speculate that upon hypergravity exposure cells experience an increase in ROS production, to which some respond activating a cell-death program. In turn, increases in cell death may influence the cellular homeostasis of the animal, inducing premature cell differentiation and perturbing activation of neoblasts and their subsequent accumulation to form a blastema. Further experiments will be necessary to validate our hypothesis.


Hands-on Activity Bacteria Transformation

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Summary

The Enterococcus faecalis bacterium lives in the human gut.

Engineering Connection

Bacteria are the most common organisms modified by genetic engineers due to the simple structures of bacteria cells compared to those of eukaryotic cells. Engineers are able to add genes to bacteria using recombinant plasmids, which enable the bacteria to produce the desired beneficial proteins. Students use a paper model to simulate this real-life process used by bio-technicians.

Learning Objectives

After this activity, students should be able to:

  • Model and describe the process used by engineers to modify the genome of bacteria.
  • Explain why bacteria are genetically modified more often than other organisms.
  • Discuss possible applications for genetically modified bacteria.

Educational Standards

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NGSS: Next Generation Science Standards - Science

HS-LS1-1. Construct an explanation based on evidence for how the structure of DNA determines the structure of proteins which carry out the essential functions of life through systems of specialized cells. (Grades 9 - 12)

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All cells contain genetic information in the form of DNA molecules. Genes are regions in the DNA that contain the instructions that code for the formation of proteins, which carry out most of the work of cells.

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International Technology and Engineering Educators Association - Technology
  • Medical technologies include prevention and rehabilitation, vaccines and pharmaceuticals, medical and surgical procedures, genetic engineering, and the systems within which health is protected and maintained. (Grades 9 - 12) More Details

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State Standards
Texas - Science
  • describe how techniques such as DNA fingerprinting, genetic modifications, and chromosomal analysis are used to study the genomes of organisms. (Grades 9 - 11) More Details

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Materials List

  • scissors
  • tape, 3 small pieces , one per group these represent plasmid DNA and mammal DNA containing the insulin gene helpful to print page 1 on different colored paper than page 2 , one per group , one per student
  • stapler (can be shared among groups)

Worksheets and Attachments

More Curriculum Like This

Students learn how engineers apply their understanding of DNA to manipulate specific genes to produce desired traits, and how engineers have used this practice to address current problems facing humanity. Students fill out a flow chart to list the methods to modify genes to create GMOs and example a.

As a class, students work through an example showing how DNA provides the "recipe" for making human body proteins. They see how the pattern of nucleotide bases (adenine, thymine, guanine, cytosine) forms the double helix ladder shape of DNA, and serves as the code for the steps required to make gene.

Students learn about mutations to both DNA and chromosomes, and uncontrolled changes to the genetic code. They are introduced to small-scale mutations (substitutions, deletions and insertions) and large-scale mutations (deletion duplications, inversions, insertions, translocations and nondisjunction.

Students reinforce their knowledge that DNA is the genetic material for all living things by modeling it using toothpicks and gumdrops that represent the four biochemicals (adenine, thiamine, guanine, and cytosine) that pair with each other in a specific pattern, making a double helix. Student teams.

Pre-Req Knowledge

A basic understanding of protein synthesis and DNA's role in the cell/body is helpful so students can follow how changes in DNA result in major changes in the characteristics of organisms.

Introduction/Motivation

How big are bacteria? (Answer: most are only a few micrometers. Micrometers are one millionth of a meter. 1 cm = 10,000 micrometers 1 mm = 1,000 micrometers) Bacteria are so small that most are less than one-tenth of the diameter of a human hair.

How many different species of bacteria exist? (Answer: Estimates vary from 10 million to 1 billion species.) So far, we have only discovered less than 10,000 different species, but some scientists estimate that as many as 1 billion different species of bacteria may exist on Earth! How prolific are bacteria? Bacteria are so plentiful that a 20-ounce bottle of water may contain up to 600 million bacteria!

Bacteria are everywhere, and most of the time they are harmless. In fact, many are beneficial to people because they are useful and necessary to a healthy human body and environment. What are some examples of these "good" bacteria? (Possible answers: E.coli within the intestines of mammals, bacteria within the soil, bacteria used to make foods such as yogurt.) Without bacteria, we would not be able to digest food or produce some of our favorite foods such as yogurt and cheese. The bacteria we might generally call "bad" for us include those responsible for causing illnesses like food poisoning.

Do you think genetic engineers could use and modify bacteria for any purpose? (Answer: Yes) Bacteria are the most commonly modified organisms. Let's look at how we can modify these bacteria and why we would want to modify them.

Procedure

Bacteria can be adapted to produce a number of useful materials. Because of the simple structures of bacterial cells, they are the most commonly modified organisms. Many times, and in this activity, a gene is simply added to the bacteria, causing the bacteria to be able to produce a useful protein, such as insulin. Other changes to bacteria can reconfigure the cellular respiration product to create desirable byproducts such as diesel or plastic molecules instead of the usual byproduct, such as carbon dioxide.

The common method used for genetically modifying bacteria is to use recombinant plasmids. Plasmids are circular pieces of DNA when placed near bacteria, the plasmid is absorbed and incorporated into the bacterial cell. Once inside the bacteria, the plasmid is treated the same as the bacteria's original DNA. This means that the bacteria will use this new DNA from the plasmid to create proteins, and the plasmid will be replicated when the cell divides.

The process of creating genetically modified bacteria used in this activity is one of the simplest methods. First, a desired gene must be selected from some (any) organism, that is, the gene that codes for the creation of insulin protein, and removed from the DNA of that organism. Genes are removed using restriction enzymes. These enzymes search for specific nucleotide sequences in the DNA, called recognition sites, where they "cut" the DNA by breaking certain bonds. When the bonds are broken in a staggered manner it creates "sticky ends." If the enzyme breaks the bond to create a straight cut, the ends are called "blunt ends." The next step is to use the same restriction enzyme to cut open the plasmid.

The isolated gene is now placed where the plasmid was cut, and they are bonded together using another enzyme called ligase. Now a recombinant plasmid has been produced. The final step is to get the plasmid into a bacteria cell. Sometimes simply placing the bacteria in the correct environment is enough to artificially induce the bacteria to intake the recombinant DNA, otherwise some special method may be required if the plasmid is too large to cross the bacteria's cell membrane. Figure 1.shows a diagram of the entire process. Figure 1. The process to build a recombinant plasmid to modify bacteria.

  • Gather materials and make copies of the Modeling Bacteria Transformation Worksheet, one per group, and Assessment Questions, one per person.
  • Make copies of the DNA Sequences for Cut-Outs, one per group it is helpful if the plasmid DNAs (page 1) are printed on different colored paper from the mammal DNAs (page 2) to help distinguish them during the activity. Then cut the DNA sequences into strips.
  • If time is short, tape the cut-out plasmid DNA into circles with the DNA sequence facing outward. Otherwise, have students do this in step 2.

  1. Divide the class into groups of two students each. Hand out to each group a worksheet and its two strips of DNA sequences, pointing out which will be used to create the plasmid, and which is the mammal DNA containing the insulin gene.
  2. Direct groups to tape together the ends of the plasmid DNA to form a circular piece of DNA (see Figure 2) so that the printed sequence is visible on the outside of the plasmid. This is the initial plasmid that will be modified with the insulin gene. Figure 2. Illustration of the circular DNA created in step 2.