Using a novel approach — recombineering — to generate <italic>odf2</italic> null alleles

doi:10.1007/s11515-011-1176-9

Front Biol

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Issue () : 83-91 https://doi.org/10.1007/s11515-011-1176-9

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Using a novel approach — recombineering — to generate odf2 null alleles

Rongsun PU(

)

School of Natural Sciences, Kean University, Union, NJ 07083, USA

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Abstract

This article uses a real-life example to illustrate the concept and methodology of recombineering, a revolutionary genetic engineering technique based on phage-mediated homologous recombination. A step-by-step approach is presented along with a flow diagram, from obtaining gene-harboring BACs to the in vitro generation of a conditional null allele. This method can be used to target any gene at any position, without the knowledge or use of any restriction site. The extensive applicability of recombineering to gene manipulation is discussed.

Keywords gene manipulation recombineering odf2

Corresponding Author(s): PU Rongsun,Email:rpu@kean.edu

Issue Date: 01 February 2012

Cite this article:

Rongsun PU. Using a novel approach — recombineering — to generate odf2 null alleles[J]. Front Biol, 0, (): 83-91.

URL:

https://academic.hep.com.cn/fib/EN/10.1007/s11515-011-1176-9
https://academic.hep.com.cn/fib/EN/Y0/V/I/83

Fig.1 Exons 5-8 are conserved (boxed area) among variants of gene transcript, as shown in Ensembl.

Fig.2 A flow diagram summarizing the major steps involved in generating a conditional null allele using recombineering. Drawing is not to scale.

Fig.3 Targeted insertion of two loxP-PGK-gb2-neo-loxP cassettes into gene. The first cassette was to be inserted between exons 4 and 5. The second cassette was to be inserted between exons 8 and 9. Homologous recombination mediated by pRedET results in the introduction of loxP sites flanking exons 5-8. Drawing is not to scale.

Tab.1 Primer sequences for insertion of cassettes via PCR.

Tab.2 Primers for confirming the insertion of the upstream cassette at 10,700 bp of using PCR.

Fig.4 Sequence of the loxP site.

Fig.5 gene with the upstream loxP site insertion at 10700 bp. Green arrows indicate the positions of primers to verify the insertion of the loxP site using PCR.

Fig.6 Final targeting construct with three loxP sites. Two of the loxP sites flank the selectable marker/cassette that is inserted between exons 8 and 9.

Fig.7 The introduction of Cre recombinase can result in three different constructs, one of which can be further manipulated to generate a conditional null allele. (a) Complete excision, resulting in an null allele; (b) Type I excision, resulting in retention of the cassette; (c) Type II excision, resulting in two loxP sites flanking exons 5-8. The conditional null allele can be generated by introducing Cre recombinase again and inducing its expression. This leads to the deletion of exons 5-8.

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