Gene Knockout

Gene targeting technologies are used to modify genomes of any living organismsWhen a mutation inactivates a gene function it is called as gene knockout. Gene knockout methods are used for the identification of a specific gene function by inhibiting the function of the particular gene. Gene knockout has its application both in classical genetics and modern techniques such as functional genomics. During the initial timescale, gene knockout was carried n transposon mutagenesis. The major disadvantage of that method is the laborious screening to find the knocked out gene. Knockout of other organism has been carried out using genetic engineering. In vitro techniques are used to modify genes on plasmids or Bacterial Artificial Chromosomes (BACs) and then this modified constructs is moved to the organism of interest by cell culture techniques. Few other methods use a combination of genetic engineering and in vivo homologous recombination. But this combination is not so efficient. Recombineering is the latest tools for producing gene knockouts. It directly states bacterial chromosome or to modify any plasmid or BAC in vivo as a prelude steps. This technique does not depend on restriction sites. A drug cassette can be placed anywhere within a gene or the open reading of the gene can be replaced with the drug cassette. Either way, the desired construct is selected for.

Technologies for gene-knockout

The best approach to produce a gene knockout is homologous recombination and through gene knockout methods a single gene gets deleted without effecting the all other genes in an organism. With the help of the gene knockout the organism where the gene of interest becomes inoperative is known as knockout organism.  When more than one gene is get knocked out in an organism then is called double knock out or DKO, triple knockout or TKO and quadrule knockouts or QKO depending on the number of genes.

Gene technology procedure

• Gene knockout is carried out together with elements such as plasmid, DNA construct or bacterial artificial chromosome.
• Gene knock out procedure often generate transgene animals where the target gene has been altered. To produce transgenic animals, embryonic stem cells or ES cells get genetically modified and in following step the transformed ES cells are placed in early embryos. The transformed animals thus produce has the ability to carry forward the transformed gene in following generations

Theory

Recombineering is defined as an in vivo homologous recombination-mediated genetic engineering. The recombination is carried out by use of a bacteriophage. Bacteriophages like λ Red, RecET, or similar systems are mostly used. Recombineering can also be used for deletions, point mutations, duplications, inversions, fusions, and tags. A linear DNA substrate containing the desired change or homologies is introduced to the target DNA into cells. The cells express the phage-encoded recombinant enzymes. These enzymes aid in adding the linear DNA into the target. Thus recombinant molecules are created. The most used recombineering system is the bacteriophage λ Red system. This system consists of three proteins, Gam, Exo Beta. The Gam protein inhibits the E. coli RecBCD exonuclease, which normally degrades linear dsDNA. Gam is not absolutely required for recombineering but increases the frequency of dsDNA recombination up to 20-fold. Exo is a 5’→3’ double-strand DNA specific exonuclease and is required for dsDNA recombination. The Beta protein, a ssDNA annealing protein, is the central recombinase in recombineering. Importantly, host recombination functions, including the key recombination protein RecA, are not required for recombineering.

Equipment

• Thermo cycler for PCR
• Agarose gel apparatus
• Power supply
• Spectrophotometer to read DNA concentration at 260nm and cell density at 600nm
• Electroporator
• Constant temperature bacterial incubator set at 30°-32°C. Should contain a roller for liquid culture tubes and shelves for petri plates 32° and 42°C shaking (200rpm) water baths (42°C cannot be an air shaker)
• Low-speed centrifuge with Sorvall SA-600 rotor (or equivalent) at 4°C
• Refrigerated microcentrifuge at 4°C
• Gel imaging system
• Insulated ice bucket
• Sterile 35 to 50 ml plastic centrifuge tubes
• Sterile 50 and 125 ml (or 250) Erlenmeyer flasks, preferably baffled
• Micropipettors
• Sterile, aerosol-resistant pipettor tips
• Pipettes of various sizes
• PCR tubes (0.2 ml flat cap tubes)
• 5 ml microfuge tubes
• Sterile glass culture tubes with stainless steel closures for culturing bacteria
• Spectrophotometer cuvettes
• Electrotransformation cuvettes with 0.1cm gap (pre-chilled)
• Petri Plates – 100 x 15mm
• Optional but highly recommended: DNA analysis software

Materials

• Agarose
• DNA molecular weight markers
• Ethidium Bromide
• PCR cleanup kit such as Qiagen Qiaquick PCR purification kit
• Platinum Taq DNA Polymerase kit (Invitrogen) or similar DNA polymerase with proof reading ability
• PCR grade dNTP set
• Double distilled sterile chilled H2O
• Two 70 base primers, desalted but not further purified. See Table 1 for ~20 base drug cassette primer sequences. The ~50 base homology sequence is dependent on the construct being made.
• Primers flanking the knockout mutation and within the drug cassette for confirming the construct
• Template DNA (See Table 1)
• Recombineering-proficient cells. See Table 2 for some options. Plasmids that supply the Red functions are also available. They can be introduced into your strain of choice.

1. The melting temperature (TM) of these primer pairs is 58°-62°C. Thus, an annealing temp of 54°C will work for all of them. All primer pairs are designed to include a transcriptional promoter. For some genes, the endogenous promoter and shine delgarno sequence are strong enough that the orf can be replaced directly with the drug resistance orf.
2. Only TetA is required for tetracycline resistance. This set of tet primers makes a smaller cassette but it is unregulated.
3. Using the spectinomycin cassette to knock out genes can be tricky, with the concentration of Spec needed to allow selection and at the same time prevent background growth. This concentration must be determined for each construct and in each strain.

 Solutions & buffers

Step 4

LB (Luria Broth) pH 7.2

Add water to 1 liter and autoclave for sterility

 Step 5

TMG

Add water to 1 liter

Adjust to pH 7.4 with HCl.

Autoclave

LB Plates +/- Drug

Add 15g Bacto Agar to LB broth to make plates. The concentration of drug  needed for selection depends on whether the drug cassette will be in multi-copy (plasmids) or single-copy (BAC, PAC, chromosome).

Drug Concentrations for Plates or Broth

* Using the spectinomycin cassette to knock out genes can be tricky, with the concentration of Spec needed to allow selection and at the same time prevent background growth. This concentration must be determined for each construct and in each strain.

Protocol

Duration – including waiting two days for oligo orders, from start to confirmed knockout is about 7 days. This protocol is written assuming the recombineering will be done in E. coli K12. Some paramenters such as growth conditions and electroporator settings may vary with other bacterial species.

 Step 1 Design Construct and Order Primers

 Duration 30-60 minutes

1.1 Obtain DNA sequence of the gene or region you wish to knock out. This target sequence must be part of a replicon (BAC, PAC, plasmid, bacteriophage, chromosome) that will replicate in E. coli (or other recombineering-proficient organism).

1.2 Decide which drug-resistant cassette you wish to use for your knockout. It is helpful if the drug cassette is a different size than the replaced gene.

1.3 Using the DNA analysis software, paste the sequence of the drug cassette you choose (include promoter and transcriptional terminators as required) into the file containing your gene/region of interest exactly as you want the final construct to appear. The two novel DNA junctions you have created by this insertion step.

1.4 Design the primers. The ~70 base primers will contain, at the 5’ end, ~50 bases of homology. This is the sequence just outside the new junctions in the in silico construct you have created. In addition, the primer will also contain ~20 bases at the 3’ end that will prime synthesis of the chosen drug cassette. The primers can also include additional short sequences such as His tags, frt or lox sites, or restriction sites.  Using the formula of 4°C for a G/C base pair and 2°C for an A/T base pair, the annealing temperature of the primers (include only the drug cassette priming region of the primer) can be set to a suitable value (e.g. 60°- 64°C) by shortening or lengthening the primer length. The two primers should have similar annealing temperatures.

1.5 Order two ~70 base primers. 100nMole scale is sufficient (and normally required) for this length. Other than desalting, no additional purification is needed or wanted. The four ~20 base primers for confirming the knockouts in Step 6.1 should be designed and ordered now too. These consist of two primers in the flanking DNA and two primers in the drug cassette.

 Step 2 Set Up and Generate Linear Substrate by PCR

Overview

Using the chimeric primers ordered during Step 1 and an appropriate template, the linear recombination substrate will be made and purified.

Duration 5-10 min set up, ~3-4 hours for PCR, and 10 min for cleanup. 1 hour for gel and DNA quantitation.

2.1 Set up PCR reactions using the two ~70 base primers ordered in Step 1.  A

typical reaction is as follows:

Typical 50μl PCR reaction mix

If a plasmid is used as a PCR template, it must be linearized by restriction. To minimize residual uncut plasmid, use the least amount of linear plasmid possible for the PCR template. Remember that even when cut with a restriction enzyme, some circular plasmid will remain. These will readily transform cells to drug resistance and will show up as “false positives”.

Typical reaction conditions for a ~1.5 kb cassette using Platinum Taq are:

If linear plasmid DNA was used as a template and you are getting residual plasmid transformation from the uninduced control in Step 4.1, cut the PCR product with the modification-dependent restriction enzyme DpnI, which will cut the plasmid DNA but not the unmodified PCR product. DpnI digestion may not totally eliminate plasmid background, however. Tip It is not necessary to gel purify the DNA. In fact, exposure of the PCR product to direct ultraviolet light will damage it, and may result in abnormal recombination frequencies as well as mutations.

 2.2 Clean the PCR product following instructions on a kit such as Qiagen Qiaquick PCR purification kit. Elute in a small amount of TE at a final DNA concentration of 100ng/μl.

2.3 Run a sample of PCR product on a gel with molecular weight markers to confirm size.

2.4 Determine DNA concentration of PCR fragment.

Step 3 Preparing Cells Competent for Recombineering

Overview Cells are prepared to be recombineering-proficient and ready for electrotransformation with the linear substrate made in Step 2. Duration about 3.5 hours

Preparation The previous day, grow a 5 ml overnight culture of the chosen recombineering cells at 30-32°C.

 3.1 Dilute overnight culture by adding 0.5 ml of the overnight to 35 ml of LB medium with the appropriate drug(s) if needed, in a 125 ml (or 250 ml) baffled Erlenmeyer flask. Dilute the overnight at least 70-fold. Grow cells in a H2O bath at 32°C with shaking (200rpm) until OD600 is from 0.4-0.5 (approximately 2 hrs).

 3.2 Transfer half the culture to a 50 ml (or 125 ml) baffled Erlenmeyer flask and place that flask in a 42ºC H2O bath to shake at 200rpm; keep the other flask at 32ºC. Shake for 15 min. The culture at 42ºC is now induced for the recombination functions and the 32ºC culture is the uninduced control. Both flasks will be processed identically during the rest of the protocol.

3.3 Immediately after the 15 min induction, rapidly chill both cultures in an ice-water slurry; swirl the flasks gently. Leave on ice for 5-10 min. Label and chill the necessary number of 35-50 ml centrifuge tubes for the induced and uninduced cells.

 3.4 Transfer both the induced and uninduced cultures to the chilled centrifuge tubes and centrifuge 7 min at ~6500 x g (6700 rpm in a Sorvall SA-600 rotor) at 4ºC. Using sterile technique, aspirate or pour off supernatant.

3.5 Add 1 ml ice-cold sterile distilled H2O to the cell pellet and gently suspend cells with a large disposable pipet tip (do not vortex). After cells are well suspended, add another 30 ml of ice-cold distilled H2O to each tube, seal, and gently invert to mix, again without vortexing. Centrifuge tubes again as in previous step.

3.6 Promptly decant the 30 ml supernatant very carefully from the soft pellet in each tube and gently suspend each cell pellet in 1 ml ice-cold distilled H2O.

 3.7 Transfer the suspended cells to pre-chilled microcentrifuge tubes. Centrifuge 30 sec at maximum speed in a 4°C refrigerated microcentrifuge. Carefully aspirate supernatant and suspend cells in 200 μl sterile ice-cold distilled H2O and keep on ice until used.

 Step 4 Electrotransformation of Linear Substrates into the Recombineering-ready Cells

Overview Recombineering-proficient, electrocompetent cells from Step 3 are transformed, via electrotransformation, with the linear substrate from Step 2 to make the knockout. Duration About 2.5 hours including outgrowth

4.1 In labeled cuvettes on ice, place 50μl of electrocompetent cells. Pipette in 1 μl (~100ng) of salt-free PCR fragment. Next take a 200 μl pipette tip and pipette up and down several times to mix. The cells are now ready for electrotransformation.

 4.2 Transform the DNA into the cells by electrotransformation. The electroporator should be set to 1.8kV. For optimal results, the time constant should be greater than 5 msec, however, we have obtained recombinants with time constants as low as 4.5 msec or so. Lower time constants generally indicate impurities or salts in the cells or the DNA. Occasionally a cuvette may be defective and will arc but arcing is often a sign of too much salt.

 4.3 Immediately after electrotransformation, add 1 ml of room temperature LB medium to the cuvette. Do this before proceeding to the next electrotransformation. After all the electrotransformations are complete, transfer the 1 ml electrotransformation mixes to sterile culture tubes and incubate with shaking (or rolling) at 32ºC for two hours to allow completion of recombination and expression of the drug-resistance gene. To ensure that each recombinant is independent, after an outgrowth of 30 minutes, the cells can be plated on filters on LB plates for further outgrowth.

 Step 5 Selecting for Knockout Mutations

 Overview Dilution and plating of cells to select for the knockout mutant.

Duration ~30 minute for dilution and plating of cells then 22-24 hours for colonies to incubate

5.1 Following the outgrowth, make 10-fold serial dilutions of the experimental cultures out to 10-6 in a buffered medium lacking a carbon source such as TMG. To select recombinants, spread 0.1 ml of the undiluted culture and of the 10^-1 and 10^-2 dilutions on plates selective for the recombinant. Also assay total viable cells by plating 0.1 ml of the 10^-4, 10^-5, and 10^-6 dilutions on LB plates. A determination of cell viability allows calculation of a recombinant frequency. If the number of viable cells is too low, less than 10⁷/ml or so, recombinants may be rare or not found. For the control cultures, both the uninduced (32°C) and the induced (42ºC) to which no DNA was added, plate 0.1 ml of the undiluted culture on a single selective plate.

5.2 Incubate plates at 30-32° until medium-sized colonies appear, normally 22-24 hours.

Step 6 Confirming Knockout Mutations

 Overview Using PCR to confirm that the knockout has been made.

Duration 30 min set up, ~3-4 hours for PCR. ~1 hour for gel

6.1 For confirming a knockout by PCR, use two pairs of primers, each pair having one primer in DNA flanking the targeted region and one primer in the drug-resistant cassette, and amplify the two junctions. Another PCR reaction, using the two outside flanking primers, should also be performed to confirm the absence of the gene to be removed, thus ruling out the possibility of a duplication event. As a control, the parent cells should be used as a template. The presence of duplications can indicate that the knockout was made in an essential gene or is polar on one. The frequency of recombination gives an indication as to whether your construction has removed or is polar on an essential gene. Typical knockouts are 10⁴/10⁸ viable but for an essential gene, the frequency is typically >100-fold reduced.

6.2 Run a sample of the PCR products on a gel with molecular weight markers to confirm sizes. If all products are the expected sizes, the knockout is ready to use.

Different Methods for gene knockout

Homologous Recombination- homologous recombination is the conventional method for gene knockout and widely used in genome engineering. This method comprises of nucleotide exchange between DNA sequences which are either similar or identical. Homologous recombination method includes a DNA construct with the mutation of choice and a drug resistance cassette to be interchanged in place of knockout gene. Additionally, the construct also includes a homologous region of nearly 2Kb with the target gene. Microinjection or electroporation are the most common methods which are next applied to transfer the construct into desired organism. The incorporation of vector construct into target site depend on the DNA repair mechanism of the organism. Once incorporated the vector construct will result in alternation of wild ype gene and eventually production of non-functional protein. However the efficiency of homologous recombination accounts only upto 10⁻² to 10^-3 integration of DNA.

Site-Specific Nucleases- There are namely three methods, zinc fingers, TALENS and CRISPER which is known to introduce double stranded breaks in DNA. Following DNA damage, the cells own repair mechanism get functional through non-homologous end joining (NHEJ), to ligate two open ends.  The repair mechanism being finished imperfectly generates insertion or deletion mutation which results in frame shift mutation. Following the mutation the gene produces non-functional protein and generates a knockout for the gene of interest.

Zinc-Fingers

Zinc finger nucleases (ZFNs) are restriction enzymes widely used in genome engineering for initiating double stranded breaks. They are known to act as site specific endonuclease which has the ability to bind to DNA at a specific site. Zinc finger nucleases (ZFNs) contain two part, a zinc finger DNA binding domain fused with a DNA cleavage part. The DNA bonding domain is consisting of 3-6 zinc finger repeats which can further recognize and bind 9-18 base pairs in a specific DNA sequence. On the other hand the cleavage domain is comprised of a type II restriction endonuclease FokI. When both the domains are assembled together, the zinc finger endonuclease protein acts as a highly effective genomic scissors. When delivered in cell, Zinc finger nucleases (ZFNs) are producing site specific DNA double strand break followed by homologous recombination. As ZFN plasmids are able to express ZFN and  effectively target a double stranded break, they provide an excellent opportunity to predesigned therapeutic constructs on a preselected location in genome.  ZFNs provides several benefits in targeting genome editing, such as-

• ZFNs are able to integrate or disrupt any location of genome rapidly.
• Mutations created by ZFNs are permanent and can be inherited.
• Genome editing are possible with the help of single transfection.
• They can be used for a wide range of mammalian cell lines
• No antibiotic selections are required for positive clone selection
• Knock-out/ knock-in effects are seen to exists nearly for two months

Applications-

• Zinc finger nucleases (ZFNs) are used for creating complete knockout in several cell lines.
• They can be used in cell based screening methods by generating knock-in cells lines where endogenous target genes can be tagged with promoter, reporter or fusion tag proteins.
• They are also useful for generating cell lines to produce specific protein or antibodies in higher amount.

Although Zinc finger nucleases (ZFNs) shows several promising effects, but there are also chance of having potential side effects. When the ZFN construct do not target or not specific off target cleavage takes place. In such scenarios DNA repair mechanism cannot control the overproduction of double stranded breaks and consequently chromosomal rearrangement or cell death takes place. Off-target cleavage also generates random integration in the host genome increasing the risk of immunological response developed by cells in response to therapeutic agents.

TALENS

TALEN or Transcription Activator-Like Effector Nucleases (TALENs) are enzymes which are genetically engineered to edit gene. These enzymes are basically isolated from bacteria of Xanthomonas species and in bacteria they participate in binding and activating the host promoter. In a similarity to ZFNs, TALENS also consisting of two groups fusing ranscription activator-like (TAL) proteins with Fok1 nuclease.  TAL protein consisting of repeating motifs composed of 33to 34 amino acids which can strongly recognize nucleotides of interest.  They are also known as Repeat Variable Diresidue (RVD) due to their variable nature. As in TALEN there exist  direct relation within DNA and amino acid recognition, it is possible to engineered a specific domain to bind DNA by changing the combination of repeating motif with specific RVD. By fusing TAL with FokI , it can effectively cutting the genome. In presence of two domain of TALEN, FokI create a double stranded break.  After constructing TALEN construct they are transfected to host cell with the help of plasmid. Inside the host cell the construct get expressed and get access to the genome by entering into nucleus. Additionally, in cells TALENs are also be transferred as mRNA which do not require genomic integration process. In comparison to Zinc finger nucleases (ZFNs) TALENS shows advantages, such as-

• The design of TALEN is much easy in comparison to ZFNs. Many TAL repeats can be created with RVD code which will bind strongly with the genomic DNA with high affinity.
• The amount of TALE repeats can be extended based on desired length.

Applications-

• TALENs are used widely for plant genome modification .
• They are useful for the production of biofuels.
• They have been used to generate knockout in organisms such as zebrafish, rat , mice or c.elegance
• It also been used to treat genetic diseases such as xeroderma pigmentation ot sickle cell .

However like ZFNs, TALENs also display off target effect. The offtarget effect will eventually generate chromosomal rearrangement due to presence of unwanted breaks in DNA double strands.

CRISPER/cas9

CRISPER/cas9 is a rapid genome editing methods which is used to delete or modify specific sequences of DNA. CRSIPER is known as Clustered Regularly Interspaced Short Palindromic Repeats which can be found naturally in certain types of bacteria.  While invaded by phage viruses, bacteria use CRIPER/Cas9 method to cut and disintegrate the viral DNA. In bacteria there exists three types of CRISPER method, among them type II is most widely studied. At this method, once cut into small parts, the invading DNA gets incorporated into CRIPER locus. Once transcribed, small repeats RNA or crRNA/ CRISPER RNAs are generated. crRNA next get joined by another noncoding RNA known as trans-activating CRISPR RNA or tracrRNA and activates the endonuclease Cas9 to target the invading viral DNA. crRNA together with tracrRNA forms a single guide RNA or sgRNA .CRISPER/Cas9 generates DNA double stranded breaks which can be repaired by two mechanism-i) nonhomologous end joining  or NHEJ, where homologous double strands are absent and ii)  homology-directed repair which is occurred in presence of synthetic DNA repair template.

Applications-

• CRISPER/Cas9 gene editing system is useful for Homology-directed repair (HDR) mechanism.
• They can be used for gene silencing
• They are functional for transiently activate endogenous genes.
• It is useful for DNA free gene editing methods.
• They are used for transiently silenced expression of genes.
• Preparation of transgenic animals as well as embryonic stem cells.

Following the discovery of CRISPER/Cas9 system, it has been extensively used in several organisms for gene targeting such as plants, humans, c.elegance, zebra fish, yeast, drosophila or mouse. They are now days used to produce single point mutations in presence of single gRNA.   In comparison to TALENs and ZNFs, CRISPER/Cas9 is easy to use as here only crRNA required redesigning to target any gene specifically. Additionally, this method also enables genome wide analysis of gene function by producing libraries of large gRNA.

 What is Gene knock in?

In genetic engineering, gene knock in refers to insertion or one to ne substitution of any locus which is normally absent in the target organism. By this method any gene, exon with mutation or tag can get inserter or knocked in and they are mostly performed in mice.  Commonly knock-in method is applied to create a model organism to study functional aspects of a specific disease. Here the exogenous gene also gets added through the mechanism of homologous recombination. To perform a gene knock-in following steps are performed-i) a mutated or reporter gene is first cloned into a vector. ii) These genes contain a flanking region known as loxP which are able to go an inverse recombination with a cre recombinase enzyme which is present in the target site. Iii)  Recombination of loxP with cre recombinase takes out the intervening target DNA and instead the mutated or reporter gene gets introduced in the target site. iii) Embryonic stem cells having the modified gene next placed into early mouse embryo cavity following transfer into a surrogate female mouse. .iv)  Further the mouse embryo developed to produce chimeric mouse having the germ line expression of the mutated or reported gene. The transgene get expressed in following generations also. These methods do not show random integration in target genome and rather gives tissue specific expression of the transgene.

Applications-

Knock-in strategy is useful in areas, such as-

• Modelling human disease- Knock-in strategies are used for the generation of skin disease like mice with mutant keratin 10(K10) gene resembles human patients where epidermolytic hyperkeratosis are seen. Knock-in mice are also generated for studying Huntington’s disease, which is a hereditary condition resulting in brain cell degeneration.
• therapeutic compounds safety and efficacy- Preclinical use of wild type mice are prohibited for the tasting of efficacy of therapeutic compounds. To combat the problem mice model of certain human  diseases are used.

Advantage of knock-in methods- Knock-in methods shows several advantages, such as-

• to mimic a disease state of human gene of interest get replaced by a mutated form.
• Integration sites can be identified easily.
• Both conditional and constitutive expression of transgene is possible
• The method is useful for the study of genes where the function has been changed.

However, knock-in methods show some limitations, such as-

• There can be presence of unexplained phenotype due to complex interaction of inserted gene with other genomic regions generating side effects.
• Forced gene overexpression generates complex pattern of protein-protein interactions.
• Knock-in mouse do not show complete resemblance to human disease state.
• Additionally generation of Knock-in mice is expensive.

Conditional gene knockout

 Conditional knockouts are performed to delete a gene in a specific tissue in specific time. This method is required for the functional study of individual gene at living organism. In comparison to gene knockout, conditional knockouts are created at adult animals rather than in embryonic stage where a mutation can show lethal effect. In mammalian cell conditional knock outs are created through homologous recombination  and following strategies are used.

Recombination with Cre-loxP –  cre or cyclization recombination enzyme is able to recognize and cut DNA sequence specifically which is further followed by recombination with second enzyme loxP.Within DNA, Cre recombinase is able to bind with two loxP site resulting a recombination between. Depending on the arrangement of loxP site, the recombination produces deletion or inversions of the target genes. Due to the inducible nature of  cre-loxP, the knockout can be induced by chemicals such as tetracyclin and  tamoxifane. Tetracyclin function in activating the cre recombinase transcription whereas, tamoxifane is responsible for nuclear transport.

Applications

Conditional gene becomes widely used methods to study human disease in different mammal model such as cancer.  Genes for breast cancer like BRCA1 has been studied through gene knockout mice with BRCA1 deletion in tissues of mammary gland to confirm the tumour suppression role.  Conditional mouse models are favoured for the study of human disease as both shows resemblance in phenotype while specific genes are deleted.

 RNAi mediated gene silencing

 RNAi or RNA intereference is a posttranslational modification which is initiated by double stranded  RNAs ( dsRNA). At the time of RNA intereference, double stranded RNAs are cut out or ‘diced’ out by an enzyme Dicer, member of RNase III family into smaller parts and those small fragments are known as interfering RNA or siRNA.  Short siRNA sequences are composed of two strands namely a guide strand and a passenger strand. Following generation of small siRNA fragments, they get attached with the special protein called Argonaute protein.  Argonaute 2 or Ago 2 is an endonuclease which is responsible for unwinding of guide strand and a passenger strand. Once the double strands are separated, the guide strand gets attached with RNA Interference Specificity Complex or RISC. RISC altogether with guide strand attached to mRNA with a complementary sequence and results in endonucleolytic cleavage degrading target mRNA. SiRNA are transferred to target cells by transfection agents such as cationic lipid or polymer based or electroporation.

Transfection- before beginning of transfection process, individual siRNAs are designed against the target gene of interest. As a delivery reagent, nanoparticles or cationic liposomes are used which can directly transfer the siRNA construct inside a cell. As a commercially available transfection reagents like lipofectamines are widely used.

Electroporation- To deliver siRNA inside the cell, electric pulsed are used. Cellular membrane being made of phospholipids is susceptible to electric pulses. In an occasion of quick but powerful electric pules, lipid molecule get reoriented and produce hydrophilic pores. This leads to the uptake of nucleic acids or molecular probes.

Viral delivery-Several  recombinant viral vectors such as retrovirus, adeno-associated virus, adenovirus or  lentivirus are used for delivery of siRNA. Among the viral vectors, lentivirus being able to stable delivery of siRNA into target cells are the popular choice as they can be delivered in transduced dividing cell or in cell nucleus. Lentiviral vectors are also able to bond to shRNA into genome allowing stable siRNA to produce longer knock down effect.

Although RNAi mechanism shows gene known down with target specificity, however off target effect r knock down of unintended genes may take place. To reduce such off target effect several strategies has been identified, such as-siRNAs are chemically modified to preferentially induce the transfer of guide strand to RISC complex or use of pool of siRNA to target single gene to reduce the effect of gene targets which are non-specific and shows off target phenotypes.

Applications

To analyse the function of individual gene, siRNAs are studied in several cellular mechanisms such as apoptosis, insulin signalling or cytokinesis. They have also been used to novel pathways identification and target validation in diseases such as cancer, hepatitis or HIV. Additionally in vivo application of RNAi  has also applied in animal disease model for the verification of specific targets so they can be used to develop therapeutic agents.

Advantages

• As a natural pathway RNA interference has the potential to produce therapeutic agents for diseases.
• They are able to bind and target any protein which eliminates the need of specific class of protein targeting in certain diseases
• RNAi can selectively suppress a target protein in cultured cells.
• The extent of gene silencing altogether with timing can get controlled.

Disadvantages

• siRNA molecules are less stable, have lesser half life and can supress the function of a gene silencing.
• Being highly specific, off target effect are also seen sometimes due to similarities kin nucleic acids.
• Silencing with siRNA at a transcript level might not affect the pre-existing proteins.

Frequenty asked questions

• What is the minimum length required for the homologous recombination?

For homologous recombination the recommended length is 2 Kb on each arm of the target gene. To have a successful recombination a total of 6 to 10Kb of DNA is highly desierable.

• Can PCR be used for the generation of gene targeting vectors?

PCR methods have been successfully applied for the generation of knockout vectors. For homologous recombination, high fidelity DNA polymerase is required with an error rate of of 0.3 bp per 10 kb of DNA.

• Can cloned genomic DNA be used for the generation of gene targeting vector?

In comparison to PCR from genomic DNA, PCR from cloned genomic DNA will produce fewer amounts of errors.

• What can be done to maximize the outcome of genetic knockout

There are several factors which can influence the outcome of knockout procedure. Among them some factors can be improves to get an overall higher rate of  knockout, such as –i) Choice of ES cells lines which is known to produce gene targeting with higher rate of success. ii)  Use of genomic clones which can be matched strain of cell which generated the ES cells. Iii) the length of homologous region required to be in the rage of 6-10 Kb.

• Why ES positive cells are screened?

After transformation, the positive ES clones get screened to distinguish between random insertion and homologous recombination.  For the purpose of conditional knockout, LoxP insertion site in the genome can be verified by ES cell screening.

• Methodology for positive clones of ES cells

Mostly PCR and southern blots are used for positive ES clones.  To perform PCR, primers are designed  beteen homologous recombination and antibiotic resisanc eassette. Whereas to confirm them presence of positive ES cells, the probe for southern blot are generated outside the homologues region.