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Q. What is the required minimum length of homologous region?
A minimum of 2 kb on each homologous arm is recommended. Total of 6-10 kb homologous genomic DNA should give reasonably satisfactory result. If your gene targeting facility has different requirements, we can make the design accordingly to accommodate the requirements.

Q. Is PCR method acceptable for generating gene targeting vector?
Many knockout vectors have been made successfully with PCR method. Theoretically, high-fidelity DNA polymerase gives on average an error of 0.3 bp per 10 kb, which is acceptable for homologous recombination purpose.

Q. Is cloned genomic DNA required for generating gene targeting vector?
Yes. PCR from cloned genomic DNA is preferred, because it will generate fewer errors than PCR directly from genomic DNA, which requires more rounds of amplification.

Q. Do I need BAC library screening?
You do not need BAC library screening if you use an ES cell line derived from mouse C57BL6 strain. End sequences of C57BL6 BAC library are readily available on NCBI web site. Using the end sequence information, we can map out C57BL6 BAC clones containing the gene of interest and order the clones directly. If you would like to use ES cell lines from other mouse strains, you need to screen the corresponding genomic library to identify the clones first. We provide mouse BAC library screening service for some of the mouse strains.

Q. What can I do to maximize a successful gene targeting outcome?
Many factors affect the frequency of homologous recombination, including intrinsic character of the targeted region. However, some of following steps can improve the chance of getting a successful gene-targeting result: (1) choose an ES cell line that is known to have a high success rate in your gene-targeting facility, (2) use genomic clones isolated from the matching strain of the ES cell line, (3) make the length of homologous region 6 – 10 kb,

Q. What is the purpose of screening for positive ES clones?
The purpose of screening for positive ES clones is primarily to distinguish between two events: the desired homologous recombination and random insertion, in which the vector DNA is randomly inserted into the genome. In case of a conditional knockout, a good screening approach should also be able to tell whether the distal loxP site has been incorporated into the genome or not.

Q. How to screen for positive ES clones?
There are two ways to screen for positive ES clones: 1) PCR, 2) Southern blot. With the PCR method, one primer is designed outside the homologous region, and the other primer is from Neo cassette. The PCR product should span across the shorter homologous arm. Although the PCR method is easy to perform, it has shortcomings. First, it lacks a “real” positive control. Second, PCR a large fragment from genomic DNA is not easy. In practice, the PCR method is often perform as a pre-screening method before Southern blot screening is performed.
Southern blot is the recommended screening method. The probe should be made outside homologous region. If homologous recombination has occurred, the probe should detect a band that is different in size from the band in the normal genome.

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