Identifying Gene-Trap Insertion Sites by Inverse PCR

Identifying Gene-Trap Insertion Sites by Inverse PCR Laurent Vergnes and Karen Reue ES cell lines carrying gene-trap insertions are typically charac...
Author: Andrea Hall
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Identifying Gene-Trap Insertion Sites by Inverse PCR

Laurent Vergnes and Karen Reue

ES cell lines carrying gene-trap insertions are typically characterized by screening mRNA sequences to identify the gene into which vector insertion has occurred. However, the precise integration site within the genome (typically within intronic sequence) is not revealed by this method, but is useful to know for the design of genotyping assays for mice derived from gene-trap ES cells. Below we describe an inverse PCR strategy to determine genomic sequence directly downstream of the insertion site for the gene-trap vector pGT1Lxf, one vector used in the BayGenomics gene-trapping project. Because additional vectors have also been used to generate BayGenomics ES cell lines, the primers and their localization described here should be confirmed for the particular gene-trap vector used. Inverse PCR allows the amplification of DNA segments that lie outside of known sequence boundaries (Fig. 1). Genomic DNA is first digested into small fragments with a restriction enzyme that cleaves at frequent intervals and digests the gene-trap vector near one end. The resulting fragments are diluted and ligated under conditions that favor intramolecular circularization of single fragments. The sequence located at one end of the gene-trap vector and extending downstream into genomic sequence is then selectively amplified using PCR primers derived from the gene-trap vector sequences (in inverse orientation), such that the genomic sequence contained in the circularized fragment will also be amplified. The resulting PCR product is amplified a second time using nested primers, purified, and sequenced. Although inverse PCR is a robust technique, difficulties can arise in its application to establish gene-trap insertion sites because of random ‘chewing’ of the vector ends prior to insertion into genomic DNA. In our experience, this typically results in a deletion of 500-1500 bp from the ends of integrated vectors. We therefore utilize a two-step approach to determine vector integration site. The first step is a set of standard PCR reactions to assess the boundary of intact vector ends in the specific ES cell line/mouse strain of interest. The second step is inverse PCR using primers selected for their localization near the end of the intact vector sequence, as determined in step 1. Although in theory the 5’ and 3’ ends of the vector could be used with similar success, we have designed this protocol based on 3’ end sequences of pGT1Lxf because the 5’ end contains sequences that are also present in the mouse genome (the Engrailed 2 gene), preventing the design of primers that are unique to vector sequences. It should also be noted that no primers sitting in the extreme 3’ 150 bp of the vector have been used, since this sequence is repeated elsewhere in the vector. Protocol (1) Prepare genomic DNA from an ES cell line or an F1 mouse carrying the gene-trap insertion. (2) Assess vector integrity: The integrity of the vector ends is assessed via standard PCR using the forward primer pGTCF in combination with each of three reverse primers: pGTCR1, pGTCR2 and pGTCR3 (see Fig. 2). Suggested PCR conditions are given at the end of the protocol section. The results of these amplifications will indicate the extent to which vector ends have been deleted prior to insertion, and will dictate which restriction digest and primer set to use subsequently for the inverse PCR. The inverse PCR primer set is selected to allow production of the smallest PCR product, as this increases efficiency and recovery. For example, if the amplification with pGTCF/pGTCR3 is positive, it indicates that the extreme 3’ end of the vector is intact, and primer set B should be used for inverse

PCR. However, if amplification with pGTCF/pGTCR3 is negative but with pGTCF/pGTCR1 or pGTCR2 is positive, then primer set A should be used for the inverse PCR. (3) Restriction digestion: Based on results in (2) above, select the appropriate primer set to be used for inverse PCR. For primer set A, samples will be digested with BfaI; for primer set B, separate aliquots will be digested with MspI and with NlaIII (use of both enzymes in parallel increases the chances of having a site at an optimal position within the genomic DNA adjacent to the gene-trap insertion site). Digest 1 µg of DNA in a total volume of 20 µl with the appropriate restriction enzyme. Incubate at 37°C for several hours or overnight. Inactivate the restriction enzymes by heating 20 min at 65°C. (4) DNA ligation: Purify DNA away from the restriction enzyme using QIAquick PCR purification Kit (or similar treatment), and resuspend in 30 µl EB buffer (Qiagen). Ligate DNA fragments with Rapid DNA Ligation Kit (Boehringer) using 2 µl DNA into a final volume of 20 µl. After ligation at room temperature for 30 min, heat inactivate the ligase for 10 min at 65°C. (5) Inverse PCR: Perform a PCR amplification using 20 ng ligation reaction and primers INVF1/INVR1 if you digested with BfaI or primers INVF3/INVR3 if you digested with either NlaIII or MspI. Analyze products by agarose gel electophoresis, keeping a small aliquot of product in reserve in case a second round of PCR is required (see below). Purify products from agarose gel (QIAquick Gel Extraction Kit). If no band is obtained, use a dilution of the aliquot of the PCR product you kept to perform a second (nested) PCR. Use a small amount (~20 ng) for a second round of PCR with the nested primers, INVF2/INVR2 or INVF4/INVR4. After the second PCR, purify products by agarose gel electrophoresis as before. (6) Sequence inverse PCR products: The PCR products can then be sequenced directly using the same PCR primers used in the final amplification and a standard cycle sequencing protocol. The sequence obtained should correspond to the gene-trap vector and extend into the genomic sequence that resides immediately downstream, thus allowing determination of the precise point of vector integration. (7) Design a genotyping assay: Once the integration site is known, it is possible to design a genotyping assay that allows unambiguous determination of all three genotypes (homozygous wild-type, heterozygous, and homozygous for the gene-trap allele). Primers that flank the integration site allow detection of the wild-type allele, whereas primers within the vector sequence can be used to detect the gene-trap allele (Fig. 3). PCR conditions are as follows:

Primer sequence:

3 min at 92°C

pGTCF, GGTCTGACGCTCAGTGGAACG

40 sec at 92°C

pGTCR1, GGGATCATGTAACTCGCCTTG

40 sec at 63°C

20 cycles

pGTCR2, GAAGATCAGTTGGGTGCACG

(-0.5°C at each cycle)

pGTCR3, CATCCCCCTTTCGCCAGCT

1-3 min at 72°C

INVF1, GTTCCCAACGATCAAGGCGAG

40 sec at 92°C

INVF2, TCAAGGCGAGTTACATGATCCC

40 sec at 53°C

20 cycles

INVR1, AAGCCATACCAAACGACGAGCG

1-3 min at 72°C

INVR2, CGAGCGTGACACCACGATGC

(+10 sec at each cycle)

INVF3, GTACTGAGAGTGCACCATATGC

5 min at 72°C

INVF4, GCACAGATGCGTAAGGAGAA INVR3, AGTTAAGCCAGCCCCGACACC INVR4, ACCCGCCAACACCCGCTG

NlaIII MspI

INVR3 INVF3

INVR4

MspI or NlaIII

INVF4

pGT1Lxf

Genomic DNA MspI or NlaIII Digestion

INVR3 INVF3 INVR4

INVF4

Ligation

INVR3

INVF3

First Round PCR with INVF3/INVR3 INVF4

INVR4

Nested PCR with INVF4/INVR4

Sequence

Fig.1. Diagram of inverse PCR strategy. Restriction sites (MspI and NlaIII) and primers (INVF3, INVF4, INVR3 and INVR4) shown are for the pGT1Lxf vector.

Set B

Set A BfaI

INVR2

pGTCF

NlaIII MspI

INVR1 INVF1 INVF2

pGTCR1

INVR3 INVF3

INVR4

INVF4

pGTCR2

pGTCR3

Fig 2. Scheme representing the 3’ end of the pGT1Lxf vector. Although several vectors used in the gene-trap ES cell lines are related and contain similar sequences, localization of the primer sequences and restriction sites should be confirmed for use with gene-traps derived from other vectors. Drawing not to scale.

Primer F

Primer R

Wild-type allele Genomic DNA Primer F

INVF

Primer R

Gene-trap allele Genomic DNA

Gene-trap vector

Genomic DNA

Fig 3. Genotyping by PCR once the integration site is known. Primers F and R are designed to amplify a ± 500 bp band in the wild-type allele. This band will not be amplified in a homozygous mutant since the presence of the vector (6-12 kb) prevents efficient amplification. Primers INVF and R are designed to detect the gene-trap allele.

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