Description

Through BAC modification/recombineering, we can place reporter genes under specific regulatory sequences of the BAC, introduce point mutations into the target gene, transfer fragments from the BAC to a plasmid vector, and add selection markers to the BAC backbone.

Notes:

a. Definition of Duration: The period from order acceptance to shipment, not including the time required for customer preparation of materials (e.g., purchasing BAC clones). 

b. Prices include the cost of purchasing BAC clones from BACPAC; the final quotation may vary depending on the complexity of the sequence introduction. 

c. This service is only applicable to BACs containing loxP sites in their backbone, most of which are available from the BACPAC resource center.

Service Description

Free BAC Modification Design

Our experienced technical team will design a detailed and efficient BAC modification plan for you based on your needs, free of charge.

Purchasing BAC for You

If you know the BAC ID you need, please provide it to us, and we will purchase it for you. If you are unsure about the BAC to use, we can help you find the appropriate BAC based on your research gene. Most BACs can be obtained from the CHORI BACPAC resource center, but some may need to be purchased from other suppliers. You can also send us Escherichia coli containing the BAC directly.

"One-Step BAC Modification"

This can be used to introduce large fragments, such as adding a reporter gene for tracing purposes behind a gene promoter, or to introduce small changes like point mutations.

Introduction of Large Fragments: The fragment contains the gene of interest (GOI) (e.g., LacZ or GFP reporter genes), flanked by an FRT site selection expression cassette with homologous arms matching the recombination region. This replaces the endogenous region on the BAC through homologous recombination. The selection expression cassette can identify successfully modified BAC clones and can be removed by Flp-mediated recombination, leaving only an FRT site.

Introduction of Point Mutations: The fragment contains the mutation region and an FRT site selection expression cassette flanked by homologous arms matching the recombination region. Through homologous recombination, this replaces the endogenous region on the BAC. Finally, the selection cassette is deleted via Flp-mediated recombination, leaving only an FRT site. The FRT site is considered a "scar" sequence because the modified BAC carries this residual FRT along with the desired point mutation. To avoid affecting gene function, this scar sequence should be placed in a non-functional region of the gene, such as an intron or UTR region.

"Two-Step BAC Modification"

The "two-step BAC modification" is used to introduce small changes (such as point mutations) into BACs without leaving FRT scar sequences. This method is suitable when there is no appropriate space near the desired mutation site to place the residual FRT scar sequence. For example, if the gene to be modified is a very large single-exon gene and the desired mutation site is located in the middle of the gene, there may be no suitable location to place a scar sequence near the point mutation.

In such cases, the two-step method should be used. This method involves positive and negative selection using a gene fragment expressing a drug selection marker (Figure 3).

1. Step One: Use homologous recombination with the gene fragment to replace the endogenous target region. Successful recombination BAC clones are obtained through positive drug selection.

2. Step Two: Use homologous recombination again to replace the drug selection gene fragment with a fragment containing the mutation site. Negative selection is then used to isolate the BAC clones that have undergone point mutation.

Adding Selection Markers to the BAC Backbone

When using such BACs to transfect cells, the selection markers on the BAC backbone will facilitate screening or tracing within the cells. This will aid in isolating cells with stably integrated BACs through drug selection.

Transferring BAC Fragments to Plasmid Vector

We can transfer any region of the BAC (up to 60 kb) to a plasmid vector for individual studies. For example, when constructing transgenic mice using the entire BAC to study the function of a specific gene on the BAC, other genes on the same BAC might influence the research results. Separating the target gene into a plasmid and using that plasmid to construct transgenic mice can avoid this issue. Additionally, from a technical standpoint, using plasmids is simpler than using BACs.

Identification of BAC Modifications

The modified BAC region will be identified through sequencing. BACs may experience fragment deletions due to the presence of repetitive sequences. To minimize this possibility, we will re-transform the modified BAC and select single clones. Multiple PCR amplifications across the entire BAC will be performed to confirm that no large fragment deletions have occurred.

Pricing and Duration

The final deliverable for this service is Escherichia coli containing the modified and validated BAC. Due to the extensive workload involved, the cost is relatively high, but outsourcing to us is more cost-effective than doing it yourself. Given the complexity and error-prone nature of the process, it might take most people up to a year to complete, requiring continual reagent procurement, learning, and troubleshooting. To better allocate and save your time, please leave the cumbersome work to us.

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