Bacterial artificial chromosome transgenic animal technology

Summary

Bacterial artificial chromosomes (BAC) are vector systems that allow the cloning of large segments of exogenous DNA. It consists mainly of a replicon composed of the redF (F-factor gene) and Ori2 sequences, as well as other regulatory sequences, and BAC replication is tightly controlled so that it remains single or low copy in Ecoli. BAC clones DNA fragments of up to 1 Mb (on average, about 200 kb). Together with the genetic stability of the F factor and the absence of deletions, recombination and chimerism, this ability to carry large insertions makes it possible to construct gene libraries using BAC. Large DNA transgenic technologies, such as the bacterial artificial chromosome (BAC), are capable of transferring multiple genes simultaneously and expressing different gene products, as well as providing remote gene expression control elements for transgenic tissue-specific expression.

Operation method

Embryo Transfer-Bacterial Artificial Chromosome Transgenic Animal Technology

Materials and Instruments

Equipment:
① centrifuge
② Incubator
③ water bath
④ small funnel, filter paper
⑤Nucleonond BAC 100 columns
⑤Nucleonond BAC 100 column ⑥Qiagen large extraction plasmid kit
⑦Spectrophotometer
⑧Pulsed field gel electrophoresis (PFGE) system
⑨ Thin-walled glass capillary tube
Reagents:
①Material: DH10B Escherichia coli
② Chloramphenicol
③10% glycerol
③10% glycerol ④Agarose medium, TBE
⑤ Restriction endonuclease, RNAase
⑥ Ethidium bromide

Move

The basic process of embryo transfer can be divided into the following steps:

I. Acquisition of BAC clones (i) Finding the desired BAC clone

1. Search for BAC clones in the Ensembl Genome database.

(1) Go to the Ensembl Genome Browser at http: //www.ensemblgenomes.org/.

(2) After entering the page, click "Vertebrates" in the upper right corner of the page to enter the search page.

(3) In the Search menu, select the gene database you want, such as "mouse" or "human". Enter the name of the gene you want, such as "H2-D1" and click "go".

(4) Under "By Feature type", select "mouse" in the database "GENE", the page will be refreshed and you can see the desired gene information, click on the gene name to enter the new page.

(5) Click "Region in detail" to link to the specific page of the desired gene, and you can see the gene cluster map around your desired gene.

(6) In the left panel, select "Configure this page". In the leftmost menu under "Sequence and assembly", select "Clones", and the related data will appear on the right side. Under "Enable/disable all External data", select "BAC map m38", click on the box in front of it, select "Labels.", and then press "V" graphic symbol (which means Save and Save) in the upper right corner of the window. Click on the square in front of you, select "Labels.", press the "V" graphic symbol in the upper right corner of the window (which means "Save and close"), and after the page is refreshed, the BACs will be listed under "Region in detail.". You can find the following BAC carriers: "RPCI-23-11503" "RPCI-23-331E12" "RPCI-23-336F22" "RPCI-23-431E4" "RPCI-23-446C22" "RPCI-24-273L24" "RPCI-24-338A13" "RPCI-24-338B13". RPCI-24-338B13".

(7) You can also select another BAC library, such as "CHORI29 clones m38". Click the box in front of CHORI29 clones m38, select "Labels." and press the "V" icon at the upper right corner of the window (which means "Save and close"), and then the page will be refreshed and the BACs will be listed under the "Region indetail.", and you can find "CH29-488C17" and "CH29-70J21".

(8) Usually 2-3 BAC clones are selected for transgenic research. Theoretically, the resulting BAC should contain as much of the 5' and 3' end sequences of the desired target gene as possible. This can increase the endogenous expression of the gene.

2. Find the BAC clones on the NCBI website (again using H2-D1 as an example).

(1) NCBI BAC database website: http://www.ncbi.nlm.nih.gov/clone/

(2) Input the desired BAC gene name in the input box, such as "H2-D1", and press the "search" button next to it to see the search results. If you want to narrow down the scope, you can also input the information of "library name" and "organization" at the same time.

(3) After selecting the desired gene, click on the gene name, and the page will be refreshed to show the simple information of the gene, such as "clone DBname" and "Allele Name". Click on the gene name after "Entrez Gene" to enter the gene information page.

(4) Scroll down the page, under "NCBI Reference Sequences (RefSeq)", you can find the link of the desired gene sequence under the "RefSeqs of Annotated Genomes: XXXXX" directory.

(5) If there are multiple genes in the catalog, you can compare the "Range" to determine the location and size of the genes, and then select one of the three links in "GenBank", "FASTA", and "Sequence Viewer (Graphics)" to find the desired sequence.

(ii) Exporting and storing BAC cloned DNA sequences

1. Record the BAC number of the desired gene. Clicking on the desired BAC will display the name of the BAC clone and the start position of the BAC insertion sequence on the chromosome (e.g. RPCI-23-331E12, start 35252064-end 35417643).

2. Enter the target gene sequence segment (17: 35252064-35417643) in "Location" under "Region in Detail" and click "GO". The DNA sequence in the BAC will be displayed on the page.

3. Select "export data" on the left side of the screen.

4. Determine the output format in the top window. For example, FASTA File, GenBank, EMBL Flat File, or EMBL if you are using SeqBuilder: select "EMBL" format in the menu and click "NEXT". The next window will allow you to select a hyperlink, a document, or a zip file of the document. Select "TEXT".

5. The desired TEXT format document will open in a new window. Select the document in its entirety, copy the information you need, and save it.

6. Process the BAC insertion sequence with DNA software such as DNAStar or Vector NT to prepare a DNA restriction endonucleotide profile for identification of the BAC.

(iii) Ordering BAC cloning

1. Visit http：//bacpac.chori.org/order clones.php.。

2. Enter the BAC name in the search box and eligible BACs will be listed. Specify an LB Stab or FISH verified clone. Click the "Clone(s) Verify" button.

3. If the BAC clone(s) has been identified, the "Clone Ordering Page" will open. Follow the instructions to pay for the desired BAC clone(s).

4. The BAC clone is present in DH10B Escherichia coli and is sent by puncture in agarose medium. it can be stored at 4℃ for 1 week.

5. Once the strain is obtained, it should be re-plated, the clones picked out and shaken overnight and stored in 10% glycerol at -80°C. The clones should be stored at -80°C.

(iv) Identification of BAC clones BAC DNA purification (i) Cultivation of bacteria

1. Use LB medium (2 mL) containing (15 pg/mL) chloramphenicol.

2. Take 1 mL of bacterial culture medium, centrifuge at 4 ℃, 5000 r/min, then resuspend in 10% glycerol and store at -80 ℃.

3. Use chloramphenicol LB medium (250 mL) containing (15 pg/mL) chloramphenicol and incubate overnight with shaking (37 ℃, 250 r/min).

4. Cool the medium to 4 ℃.

(ii) Preparation of reagents

1. Add RNAase to the cell suspension buffer.

2. Add filter paper to a small funnel and insert the small funnel into a Nucleonond BAC 100 column. Equilibrate the filter paper and column with 10 mL of Equilibration Buffer and allow to run dry. The filter paper prevents bacterial lysates from clogging the column.

(iii) Bacterial Lysis

1. Centrifuge the bacterial culture solution at 5000 r/min, 4 ℃ for 10 minutes, and discard the supernatant.

2. Resuspend the cells with cell suspension buffer (use 30 mL of buffer for every 250 mL of bacterial solution). If too many bacteria are added, the amount of buffer can be increased, and buffers from Macherey-Nagel, Marligen, and QiNucleobond can be used universally. Marligen and QiNucleobond buffers can be used in place of Macherey-Nagel buffer if necessary.

3. Add an equal amount of cell lysate and mix gently, do not use vortex. Allow to stand at room temperature for 5 minutes.

4. Add an equal amount of Neutralizing Solution. Gently turn up and down, not too vigorously, do not use vortex to mix. A viscous white precipitate is seen to form. Centrifuge the precipitate for 10 minutes at 15,000 r/min.

(iv) Extraction of BAC DNA

1. Pour the neutralized cell lysis product into a funnel and filter naturally by gravity. Discard the filtrate and funnel.

2. Add 30 mL of washing buffer to the column and drain naturally by gravity.

3. Add 15 mL of eluent to the column and allow the eluent to flow naturally into the collection tube using gravity.

4. Add 11 mL of isopropanol to the eluate and centrifuge at 4 ℃ and 10,000 r/min for 40 minutes (note that it should not exceed 10,000 r/min or the 50 mL tube will be easily ruptured).

5. Centrifugation produces white, fragile DNA sediments. Sometimes they are so small that they are difficult to see. Handle the BAC DNA precipitates with care because they tend to stick to the walls of the centrifuge tubes. If a large white precipitate is visible, something may have gone wrong during the purification phase (e.g., you forgot to add RNAase or the buffer has expired).

6. Resuspend the precipitates with 3 mL of 70% anhydrous ethanol and divide them equally between 2 microcentrifuge tubes. centrifuge at high speed for 10 minutes at 4°C.

7. Discard the supernatant and dry the products for 3 minutes. Do not let them dry completely or the DNA will break easily.

8. Filter 1 mL of PA buffer through a 0.2 μmol/L syringe filter. Do not filter the BAC DNA solution through this filter or the BAC DNA will stick to the filter membrane and be lost.

9. Resuspend the BAC DNA with 300 μl of filtered PA buffer. the DNA will dissolve readily in the PA buffer. If some of the precipitate does not dissolve, it may be some impurity from irregular handling during the purification process.

(v) Quantification of BAC DNA

1. Turn on the spectrophotometer and select the application program.

2. Lift the movable arm and add 1 μl of water to the base with a pipette; as an answer in the dialog box, then tap "OK" to start calibration.

3. Wipe the top and bottom of the spiking base clean with mirror paper.

4. Use a pipette to add 1 μl of buffer PA to the base and click "Blank" to calibrate.

5. Clean the dock, add 1 μl of BAC DNA solution and click "measure".

6. After measuring the sample, wipe it off with a piece of microscope paper and add 1 μl of water to the final sample with a pipette.

7. Save the data to the computer, clean the base and exit the software program.

BAC Clone Integrity Identification - Production of BAC DNA Restriction Endonucleotide Mapping

1. Digest BAC DNA (250-550 ng) with restriction endonuclease.

2. Assemble a pulsed-field gel electrophoresis (PFGE) system in a cooling chamber or connect a circulating cooler to the PFGE system.

3. Prepare a 0.8% agarose gel (note: make sure there are no air bubbles in the gel; discard the gel if it does not meet the requirements; every bubble affects the migration of BAC DNA).

4. Add ethidium bromide to the gel and 2000 mL of 0.5 times TBE to the gel electrophoresis tank.

5. Cut thin slices from the Midrange RFG Marker II agarose block with a razor blade and place them in the gel sample tank. (Note: Instead of preparing a BAC Sample DNA agar block, add the BAC Sample DNA directly to 6x DNA Sample Buffer; sample as for normal agarose gel horizontal electrophoresis).

6. Add 250-500 ng of restriction endonuclease-digested BAC DNA to each gel spiking tank, and add undigested BAC DNA to one of the tanks as a control.

7. Start pulsed gel electrophoresis. Pulsed field gel electrophoresis settings: initial switch time: 1 second, final switch time: 25 seconds, electrophoresis time: 17 hours, 6V, angle: 120° (Note: it takes a whole night to run the gel; if the current is too high, you need to check the concentration of TBE buffer because the resultant band will be deformed if the current is too high).

8. After running the gel, soak the gel in ethidium bromide solution for 30 minutes and then visualize the DNA bands under UV light (Note: Ethidium bromide is a mutagenic substance; it is important to follow the safety manual and dispose of discarded ethidium bromide in a non-hazardous manner).

9. Analysis of electrophoresis results

(1) DNA markers can produce dispersed bands for determination of DNA molecular weight. Adding too much DNA marker to the marker bath will blur the DNA bands.

(2) Add different amounts of BAC DNA to ensure clear bands. For example, with RNA-contaminated samples, the spectrophotometer will yield a higher concentration of DNA in the sample. In this case, not all samples contain enough BAC DNA and the DNA bands will be darker. The contaminated impurity RNA will also migrate through the gel without interfering with the visualization of the BACDNA fragments.

(3) Compare the actual restriction endonuclease profile with the results analyzed by the computer software. If there is no match, the same BAC can be obtained from C.H.O.R.I. again free of charge.

(4) BACDNA with accurate identification results can be stored in PA buffer at 4 °C for two years without degradation.

Recombination and Modification of the BAC Gene

The BAC gene can be easily modified by recombineering. Recombineering can easily clone large segments of DNA and can also cause specific mutations at any site in the gene. The procedure is described in Chapter 2, Section 2 of this document.

V. BAC DNA Transgenesis

1. Use 0.2 μm filtered PA buffer and dilute to 10x or 100x (optimal precision). The filtered PA buffer is used to dilute the BACDNA to 0.5 ng/μl. Store the DNA at 4 °C and do not freeze.

2. Divide the DNA into 4-6 portions of 50 μl each and place in sterile microtubes.

3. Use a pipette (WPI cal. No. TW100-4) to pull the needle of the microinjection out of the thin-walled glass capillary.

4. Place the back of the pulled needle in the resuspended BACDNA and wait 1-2 minutes for the liquid to pass through the capillary to the tip of the needle.

5. After the tip of the needle is visible through the lens, the needle is mounted on the microinjector and placed in the microinjection chamber filled with fertilized eggs.

6. Complete the microinjection of the prokaryotic nucleus. By properly preparing the BAC DNA prior to microinjection, the procedure will be smoother and will be as easy to accomplish as the plasmid transgene DNA solution.

7. It is not necessary to change the geometry of the microinjection needle for BAC DNA microinjection.

8. The survival rate of fertilized eggs after BAC DNA microinjection is less than that of plasmid DNA transgenic microinjection.

9. Screening and characterization of BAC transgenic first-born mice.

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