Gene Manipulation Flashcards

Question

What would happen if GFP encoding P element was inserted into enhancer elements *

Answer 1

Allows the expression of genes in a specific tissue or at a specific time by using a promoter driving GAL4 expression in desired cells or conditions. Overexpressing or ectopically expressing a gene to study its effects on development, behavior, or physiology. Ectopically expressed refers to the expression of a gene in a location, tissue, or cell type where it is not normally expressed

Answer 2

Most commonly used method • Only 5% or less of the treated eggs become transgenic progeny • Need to check mouse pups for DNA (by PCR or Southerns), RNA (by northerns or RT-PCR), and protein Loading…(by western or by some specific assay method) • Expression will vary in transgenic offspring: due to position effect and copy number

Answer 3

Difficult to do transgenesis in mammals so do homologous recombination instead 1) GOI inserted between homology arms + selection marker in plasmid 2) The targeting vector is introduced into mouse embryonic stem (ES) cells via electroporation. The cell’s repair machinery uses homologous recombination to integrate the vector into the genome at the target locus. 3) identify ES cells that have homologously combined with vector and inject these into embryos 4) embryos implanted into surrogate- offspring are chimeric (have normal and modified ES cells) 5) Chimeric mice are bred with wild-type mice to determine if the genetic modification has been incorporated into the germline. Offspring carrying the genetic modification are identified and further bred to establish a transgenic line.

Answer 4

Sequences that are identical to regions flanking the target gene in the mouse genome. These guide the vector to the correct genomic location.

Answer 5

used to make precise changes in the DNA of organisms. They are employed to disrupt, modify, insert, or delete specific genes

Answer 6

Zinc Finger Nucleases (ZFNs) are engineered proteins used for precise genome editing Composed of multiple zinc finger motifs, each recognizing a specific 3-base DNA sequence. Zinc fingers are arranged in tandem to recognize a longer, unique DNA sequence, providing specificity The zinc fingers of each monomer bind to their respective target sequences on opposite strands of the DNA. The DNA is cleaved at the spacer site between the two ZFN binding regions. The cell repairs the break using one of two pathways: Non-Homologous End Joining (NHEJ) , Homologous Recombination (HR)

Answer 7

are genome-editing tools designed to introduce specific double-strand breaks (DSBs) into DNA at targeted locations Composed of transcription activator-like effector (TALE) repeats, Specificity is determined by two hypervariable amino acids (the repeat-variable diresidue, RVD) within each repeat: NI recognizes A. HD recognizes C. NG recognizes T. NN recognizes G (and sometimes A). Multiple TALE repeats are assembled in tandem to recognize a specific DNA sequence. (One TALE repeat ,~33 aa long, has one RVD), need multiple TALE repeats to recognise DNA sequence. Then rest of process same as ZFN (cut at spacer region, repair)

Answer 8

Components of Cas9 Protein: A nuclease that cuts DNA, creating DSBs. Guided to the target sequence by an RNA molecule. gRNA combines: - CRISPR RNA (crRNA): Contains a 20-nucleotide sequence complementary to the target DNA. - Trans-activating CRISPR RNA (tracrRNA): Binds to the Cas9 protein and stabilizes the complex. sgRNA guides the Cas9 protein to the target DNA sequence through complementary base pairing. The Cas9 protein binds to the DNA only if a PAM sequence is adjacent to the target site. Cas9-sgRNA complex binds to the target DNA, and the sgRNA hybridizes with the complementary DNA strand. This forms an RNA-DNA duplex and opens the DNA double helix. Then rest of process (ds break, repair via either of the two pathways)

Answer 9

Recognition (REC) Lobe: Responsible for recognizing and binding to the guide RNA (gRNA) and the target DNA. Nuclease (NUC) Lobe: Contains the nuclease activity responsible for DNA cleavage. Composed of: HNH Domain: Cleaves the DNA strand complementary to the guide RNA. RuvC Domain: Cleaves the non-complementary DNA strand (opposite to the guide RNA). PAM-Interacting Domain: Recognizes the protospacer adjacent motif (PAM) sequence, essential for target DNA binding.

Answer 10

produces only single-strand breaks, reducing the risk of large-scale genomic disruptions If two single strand nicks are near to eachother = considered a ds break (Ignore article )

Answer 11

It is located immediately adjacent to the target DNA sequence that the Cas9 protein will bind and cut. The presence of a PAM is required for the Cas9 protein to recognize and cleave the target DNA

Answer 12

DNA repair mechanism that cells use to fix double-strand breaks (DSBs) in DNA. It relies on the presence of a homologous DNA template to guide the repair process, ensuring accurate restoration of the DNA sequence at the break site. HDR is a critical pathway for maintaining genome stability

Answer 13

is a DNA repair mechanism that fixes double-strand breaks (DSBs) in DNA without requiring a homologous template. It is an error-prone but fast and efficient pathway lack of a template often leads to mutations (indels) at the repair site which can disrupt gene function by introducing frameshift mutations or premature stop codons.

Answer 14

The system helps bacteria recognize and neutralize these viral threats by "remembering" previous infections through a form of genetic memory and using it to target and cut the phage DNA during subsequent infections When a bacterium is infected by a bacteriophage, the CRISPR-Cas system can capture a short segment of the viral DNA, called a spacer sequence. This sequence is inserted into the bacterial CRISPR array, which is a repetitive DNA region in the bacterial genome. Each new spacer sequence represents a "memory" of the virus that the bacteria has encountered CRISPR array is transcribed into a long RNA molecule, which is then processed into smaller RNA fragments known as crRNAs. Each crRNA contains a sequence (called the spacer sequence) that is complementary to a corresponding segment of the viral genome the bacterium has previously encountered crRNA guides the Cas proteins (e.g., Cas9, Csm, or Cmr complexes) to the phage DNA. The Cas proteins, guided by the crRNA, recognize the viral DNA that matches the spacer sequence and bind to it. Once the Cas proteins bind to the phage DNA, they cut the viral DNA, rendering it inactive

Answer 15

crRNA is transcribed from the bacterial CRISPR array, which consists of repetitive sequences interspersed with unique sequences (spacers) that represent viral DNA or other foreign DNA that the bacteria have previously encountered. A crRNA molecule contains a spacer sequence that is complementary to the target DNA sequence (usually within a bacteriophage genome). tracrRNA is complementary to the repeat sequence of the crRNA, and it helps bind the crRNA to Cas9. TracrRNA also facilitates the maturation of crRNA by helping to process the crRNA into smaller functional units. The tracrRNA binds to the repeat region of the crRNA, forming a duplex. This crRNA-tracrRNA complex then associates with the Cas9 protein. Cas9 is a two-domain protein that requires the binding of this RNA complex for activation. The crRNA guides Cas9 to the target DNA by base-pairing with the complementary DNA sequence, and the tracrRNA helps stabilize this interaction. The Cas9-gRNA complex (which includes both crRNA and tracrRNA) scans the DNA for a matching sequence to the crRNA spacer. Once a match is found, the PAM (Protospacer Adjacent Motif) sequence is recognized by Cas9, and the protein creates a double-strand break (DSB) in the DNA

Answer 16

refer to processes or functions that are relatively stable, consistent, and not dynamically regulated in response to immediate changes in the environment or cellular conditions

Answer 17

Investigated potential of antisense DNA strand transcription to inhibit gene activity By flipping the orientation of a gene (the HSV thymidine kinase, or TK gene) and inserting it into a vector, antisense RNA is produced. When this flipped gene is co-injected with the wild-type TK gene at a 100:1 ratio into TK-deficient mouse cells, there is a significant reduction in TK expression and cell viability compared to controls. The reduction is likely due to trans-inhibition by antisense RNA, not artifacts from DNA rearrangement. This demonstrates the potential of antisense approaches for gene regulation.

Answer 18

A process in which small RNA molecules regulate gene expression by silencing specific mRNAs, effectively reducing or preventing the production of proteins.

Answer 19

by selectively silencing or downregulating gene expression Works by introducing RNA molecules into a cell, that match the target gene's mRNA, leading to its degradation or translational repression E.g. Synthetic siRNAs (small interfering RNAs) Can be introduced by a vector

Answer 20

Presence of ds RNA initiates the process, either endo or exogenous source of dsRNA DICER cleaves the ds RNA into 21bp long fragments siRNA loaded onto RISC complex (unwound, one strand binds Argonaute = guide strand, other strand degraded) The guide strand in RISC directs the complex to complementary mRNA molecules. Base pairing occurs between the guide strand and the target mRNA Depending on the degree of complementarity, mRNA silencing occurs via: Cleavage (Degradation): If the siRNA is fully complementary to the target mRNA, Argonaute cleaves the mRNA at a specific site. Translational Repression: If there is partial complementarity, translation of the mRNA is inhibited (less common in siRNA-mediated pathways).

Answer 21

miRNAs are transcribed by RNA polymerase II (or III) as part of longer primary miRNA transcripts (pri-miRNAs) that often include hairpin structures. pri-miRNA is processed by the Drosha enzyme into a precursor miRNA, a ~70-nucleotide hairpin structure The Exportin-5/Ran-GTP complex transports the pre-miRNA from the nucleus to the cytoplasm. In the cytoplasm, Dicer, an RNase III enzyme, cleaves the pre-miRNA into a 21 nucleotide double-stranded RNA molecule, which is loaded onto RISC one strand of miRNA binds argonaute= guide RNA, other degraded miRNAs guide the RISC to target mRNAs through complementary base pairing degree of complementarity determines the silencing mechanism: perfect complementary = mRNA degraded, mismatch = translation inhibited.

Answer 22

mRNA translated in a loop (mRNA is in a loop shape) Significance: The proximity of the 5' cap and 3' poly(A) tail facilitates ribosome recycling. Ribosomes that finish translation at the stop codon can quickly reinitiate at the start codon.

Answer 23

Knock-in adds or replaces genetic sequences to study specific modifications or introduce new functionality. Knock-out completely removes or inactivates a gene to study its absence. Knock-down reduces gene expression temporarily to study partial loss-of-function effects.

Answer 24

Each well in multiplate contains cells to be treated with a specific RNAi molecule Use RNAi libraries to target specific mRNAs corresponding to the genes to be silenced.

Answer 25

They switch cuz lay lots of embryos = energetically costly for female

Answer 26

Can be used to : Knock down genes in specific tissues or cell types Study gene function at specific stages of development Perform large-scale RNAi screens to identify genes involved in specific biological processes or pathways. Knock down genes associated with human diseases in specific tissues to study their effects.