genetic modification Flashcards
(9 cards)
genetic modification methods
Homologous recombination (HR) is a precise genetic process where nucleotide sequences are exchanged between two similar or identical strands of DNA. It plays a critical role in maintaining genomic integrity by allowing cells to accurately repair double-strand breaks (DSBs), particularly during cell division. HR is most active during the S and G2 phases of the cell cycle when a sister chromatid is available as a template. During meiosis, HR also facilitates the exchange of genetic material between homologous chromosomes, contributing to genetic diversity in offspring.
In genetic engineering, HR can be harnessed to introduce targeted modifications in the genome of genetically modified (GM) cells. To track gene expression, researchers often use reporter constructs such as Green Fluorescent Protein (GFP). For example, a promoter sequence from a gene of interest can be fused upstream of the GFP coding region, forming a construct that is only expressed in cells where that gene is active—resulting in green fluorescence.
The plasmid containing GFP and the homology arms is introduced into the embryonic stem (ES) cells.
If homologous recombination occurs, the GFP gene (fused to the promoter) is inserted into the target gene’s locus, where the homology arms match the flanking sequences of the target gene.
The cells that successfully integrate the GFP (and, by extension, the target gene modification) will express GFP and glow green under fluorescence, allowing researchers to easily identify them.
However, this process generally occurs at low efficiency, particularly in mammalian somatic cells, which is why additional selection methods or nuclease-induced DSBs (e.g. via CRISPR/Cas9) are often used to enhance targeting frequency.
how to generate antibiotic resistant knockout mice
How to Generate Antibiotic-Resistant Knockout Mice
- Derivation of Pluripotent Embryonic Stem (ES) Cells
ES cells are isolated from the inner cell mass of a mouse blastocyst.
These cells are cultured on a feeder layer of mouse embryonic fibroblasts (MEFs) in the presence of leukaemia inhibitory factor (LIF) to maintain pluripotency (ability to self-renew and differentiate).
Pluripotent ES cells can:
Be injected back into blastocysts to form chimeric mice.
Be used to form embryoid bodies by adding specific growth factors.
Form teratomas in vivo, containing cell types from all three germ layers—evidence of pluripotency.
- Genetic Modification of ES Cells to Introduce Antibiotic Resistance
Linearized DNA containing the desired genetic modification and an antibiotic resistance gene (e.g., neomycin resistance) is introduced into ES cells via:
Transfection or
Electroporation (using an electric pulse to permeabilize the membrane).
The neomycin resistance gene:
Allows selection of successfully modified cells.
Is flanked by homologous sequences to the target gene, facilitating integration via homologous recombination (though random insertion can also occur).
Example (Lineage Tracing): ES cells with GFP targeted to the Brachyury gene will express GFP when the gene’s promoter is activated during mesoderm formation, helping visualize developmental processes.
- Selection of Genetically Modified ES Cells and Generation of Knockout Mice
Selection and Enrichment:
After modification, ES cells are grown in the presence of neomycin to select for antibiotic-resistant cells that contain the inserted gene.
A clonal population of ES cells with the desired gene disruption is isolated.
Creation of Chimeric Mice:
Gene knockouts are often achieved by introducing a selection cassette into the target gene in embryonic stem (ES) cells. In this process, the gene of interest is disrupted in the middle, and the GM selection cassette replaces it. If homologous recombination does not occur, and the gene is randomly integrated, the next step is to apply HSV-thymidine kinase (HSV-TK) as a negative selection marker. To ensure that only the cells with the correct modification are retained, positive selection (e.g., using neomycin to select for cells with successful gene insertion) and negative selection (e.g., using ganciclovir to kill cells with random integration) are applied. This selection process kills the cells with random integration, leaving only the targeted ES cells that have undergone homologous recombination.
After confirming successful targeting by Southern blot or PCR, the targeted ES cells are then injected into a blastocyst from a pseudopregnant mouse, which is then implanted into the mother. The offspring born from this procedure are chimeric mice, which have a mix of genetically modified (GM) cells and wild-type cells. Chimeric mice can show visible differences, such as grey and white fur. The GM cells will typically contribute to the gonads, and by breeding these chimeric mice, it is possible to produce heterozygous mice with one copy of the gene knocked out.
The blastocysts are implanted into a pseudo-pregnant foster mother to allow development to term.
Coat colour differences (e.g., agouti brown ES cells into black blastocysts) help visually assess the degree of chimerism in the offspring.
Germline Transmission:
Chimeric mice are bred with black mice.
If offspring display agouti (brown) coat colour, it indicates that the modified ES cells contributed to the germ line, confirming successful gene transmission to the next generation.
The extent of the contribution of ES cells to the formation of the chimeric mouse can be evaluated by the visual assessment of coat-colour chimerism. ES cell contribution to the germ-line can be evaluated by observing the colour of the progeny that is derived by breeding the chimeric mouse with black mice
lineage tracing
follow stem cell fate decisions, study proliferation, differentiation and migration in vivo
based on visualisation of the cells lineage via a time and spatially controlled reporter gene expression in stem/rogenitor cells that is apssed on to their cell progeny
lineage tracing of hair follicle bulge stem cells: placing the cre gene under cotrol of a promoter specific for cell population of interest. gfp expression occurs in hair follicle bulge stem cells as soon as promotor is activated in cell migration. permanent genetic modification is passed on to the progeny, independent of cre expression.
virus-mediated gene addition
Virus- Mediated Gene Addition - Viral genes are removed from the plasmid DNA and in their place exogenous DNA with its promoter is added. This plasmid DNA is then added to the vector through transfection 1. Gamma retroviruses (gRV) are convenient tools for engineering gene transfer into haematopoietic cells via integration of a DNA copy of the viral RNA into the target cell s genome and then replication in all daughter cells ǯ 2. Viral vectors are used to treat diseases such as Haemophilia B using the correct clotting gene factor IX. This allows the correct clotting factor to be secreted into the patient and effectively treating the disease. These viruses contain long terminal repeats (LTRs) and thus drive transgene expression 3. Latest trial: four of the six participants were able to stop using prophylaxis with FIX (therapeutic gene) concentrate without having spontaneous haemorrhages, even when they undertook activities that had provoked bleeding in the past. As part of virus life cycle they integrate their genome i.e. Retroviruses - Exist of protein shell containing a genome - The virus are positive strength viruses, they produce negative strand DNA using reverse transcriptase enzyme - Go from producing RNA to -DNA to +DNA
Virus uses that to integrate their DNA into the genome with an integrase and then exploit the transcription machinery of the cell. - This means their DNA will be transcribed alongside the DNA of the cell. We can use this to change our hemopoietic stem cells genetically.
reducing mutagenesis
To reduce the risk of mutagenesis during gene editing, researchers often exploit the cell’s natural double-strand break (DSB) repair mechanisms, particularly for gene correction or gene addition. DSBs are typically repaired through non-homologous end joining (NHEJ) or random integration, both of which can be error-prone. However, inducing targeted DSBs allows for more controlled genome modifications. One method to achieve this is by using zinc finger nucleases (ZFNs)—engineered proteins that can be tailored to bind virtually any DNA sequence. Each zinc finger domain recognizes a specific triplet of nucleotides, and a typical ZFN consists of multiple zinc finger domains fused to a FokI endonuclease. ZFNs can be used either to introduce mutations for gene knock-out or, when co-delivered with a donor plasmid, to mediate gene repair through homology-directed repair (HDR).
An alternative to ZFNs is transcription activator-like effector nucleases (TALENs), which function similarly but use a different DNA recognition code. Each repeat domain in TALENs recognizes a single base pair, providing a more modular and potentially more specific targeting system with reduced off-target effects. More recently, CRISPR/Cas9—a system derived from bacterial adaptive immunity—has revolutionized genome editing. It enables highly efficient and programmable introduction of mutations, precise gene correction, gene insertion, and even epigenetic modulation when fused with transcriptional or chromatin-modifying domains. These tools offer diverse applications in basic research and therapeutic development by allowing precise manipulation of the genome and epigenome.
basic principles of genome editing
what is needed?
1. molecular scissors: a nuclear enzyme to make a double stranded cut in the genome
2. homing devices: a mechanism to recognise specific dna sequences- derived from dna binding proteins such as transcription factors (ZFN, TALEN) or complementary RNA (CRISPR)
3. template: if more than a simple mutation is needed, a DNA template with homogenous arms is required to allow homology-directed repair.
crispr/cas9
Genome editing using site-directed nucleases (SDNs) involves inducing targeted double-strand breaks (DSBs) in DNA to manipulate genetic sequences. The CRISPR/Cas9 system is a widely used SDN tool due to its versatility and simplicity. After a DSB is introduced by Cas9, cells repair the break via either non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ is error-prone and often leads to insertions or deletions (INDELs), which can disrupt gene function and result in gene knock-outs. In contrast, HDR uses a supplied DNA repair template to introduce precise gene modifications or insertions, making it suitable for gene correction or replacement, though it is less efficient and typically restricted to dividing cells.
CRISPR can also be repurposed for epigenetic modifications using nuclease-defective Cas9 fused to effectors such as DNA methyltransferases (e.g., DNMT3A) or histone acetyltransferases (e.g., p300). These fusions enable targeted regulation of gene expression without altering the DNA sequence, offering powerful tools for studying gene regulation and potential epigenetic therapies.
base editing
Beyond traditional CRISPR editing, base editing enables precise single-nucleotide changes without inducing DSBs or requiring donor DNA. This approach uses a nuclease-deficient Cas9 (dCas9 or Cas9 nickase) fused to a deaminase enzyme (e.g., cytidine deaminase or adenosine deaminase) guided by gRNA. Cytosine base editors (CBEs) convert CG to TA, while adenine base editors (ABEs) convert AT to GC. These are especially useful in post-mitotic cells like fibroblasts or keratinocytes and are well-suited for in vivo delivery due to their low off-target potential and efficiency in editing without DNA breaks.
clinical applications
Gene and cell therapy are emerging as powerful clinical strategies for treating inherited skin disorders, such as recessive dystrophic epidermolysis bullosa (RDEB), which is caused by mutations in the COL7A1 gene encoding type VII collagen. In RDEB, loss or dysfunction of collagen VII leads to severe skin fragility and blistering due to impaired anchoring fibrils at the dermoepidermal junction (DEJ). Recent advances in base editing have enabled precise correction of point mutations in COL7A1 without inducing double-strand breaks, significantly reducing the risk of off-target effects. Successful adenine base editing in RDEB patient-derived skin cells has led to restoration of functional collagen VII protein at the DEJ in gene-corrected skin equivalents (RDEB=B skin), demonstrating therapeutic potential.
Moreover, induced pluripotent stem cell (iPSC)-based therapies offer a promising platform for regenerative approaches. Patient-derived iPSCs can be genetically corrected and then differentiated into fibroblasts and keratinocytes—the primary cell types involved in skin structure—under feeder-free and xeno-free conditions, ensuring clinical compatibility. These corrected and differentiated cells can be used to reconstruct functional skin grafts for transplantation, offering long-term treatment solutions. Together, these strategies represent a transformative step toward personalized regenerative medicine for genetic skin disorders.
