Homologous illegitimate random integration of foreign DNA into the X chromosome of a transgenic mouse line

Abstract

Background: It is not clear how foreign DNA molecules insert into the host genome. Recently, we have produced transgenic mice to investigate the role of the fad2 gene in the conversion of oleic acid to linoleic acid. Here we describe an integration mechanism of fad2 transgene by homologous illegitimate random integration.

Results: We confirmed that one fad2 line had a sole integration site on the X chromosome according to the inheritance patterns. Mapping of insertion sequences with thermal asymmetric interlaced and conventional PCR revealed that the foreign DNA was inserted into the XC1 region of the X chromosome by a homologous illegitimate replacement of an entire 45,556-bp endogenous genomic region, including the ovarian granulosa cell tumourigenesis-4 allele. For 5' and 3' junction sequences, there were very short (3-7 bp) common sequences in the AT-rich domains, which may mediate the recognition of the homologous arms between the transgene and the host genome. In addition, analysis of gene transcription indicated that the transgene was expressed in all tested fad2 tissues and that its transcription level in homozygous female tissues was about twice as high as in the heterozygous female (p < 0.05).

Conclusions: Taken together, the results indicated that the foreign fad2 behaved like an X-linked gene and that foreign DNA molecules were inserted into the eukaryotic genome through a homologous illegitimate random integration.

Figure 1

Transgene analysis. (A) PCR and (B) Southern blot analysis of total DNA from wild-type C57 (wt), mouse-1 (lane 1) and mouse-2 (lane 2) shows that mouse-1 was fad2 transgenic, whereas mouse-2 was nontransgenic. P, the mixture of fad2 plasmid and wt genomic DNA (A) or the 1036-bp fragments of fad2 transgene digested by EcoR I and Sca I enzymes together (B). (C) PCR analysis of total DNA from 50 heterogeneous F1 mice produced by natural mating of the C57 females and mouse-1 (male) show that all 27 F1 females (mice nos. 1-5, 15-24, 32-36, 39-41, and 44-47) were transgenic, whereas all 23 F1 males (mice nos. 6-14, 25-31, 37-38, 42-43, and 48-50) were nontransgenic. M, DNA marker. P, the mixture of fad2 plasmid and wt genomic DNA.

Figure 2

TAIL-PCR analysis of the 3' integration site. (A) Schematic diagram of the transgenic construct indicating the positions of three transgene primers (tgp) used, along with an arbitrary degenerate primer. (B) Gel analysis of TAIL-PCR products amplified from the fad2 mice. Total DNA from three fad2 mice (nos. 3, 5, and 15) was used for TAIL-PCR, and the specific amplified fragments (*) in each sample from the tertiary amplification were sequenced directly. (C) Nucleotide sequence of transgenic forward primers (underlined) tgp1 (-627 to -610), tgp2 (-381 to -360), and tgp3 (-206 to -187) and the 3' integration region in fad2 mice. The sequence from -172 to the 3' end was determined by direct sequencing. The sequencing results indicate that nucleotides from -172 to +7 were 100% identical to the transgene sequences and that the nucleotides from +1 to the 3' end were the same as the downstream sequence of the 87,507,732nd nucleotide of the XC1 region of the X chromosome. Seven nucleotides, from +1 to +7 (5'-TTAATAG-3'), were shared by the transgene and the X chromosome as a very short homologous arm. Furthermore, the sequences from -172 to -22 and -22 to +7, corresponding to the 3' end and the 5' initial transgene sequences, indicate that the foreign DNA molecules integrated as a head-to-tail array.

Figure 3

Identity of homozygous or heterozygous fad2 mice. (A) Schematic diagram of the primer positions indicating the fragments spanning the 3' integration site in wild-type C57 or transgene-specific fragments in fad2 mice. (B) Site-specific primers used to identify fad2 homozygotes and heterozygotes. C57 samples had the 495-bp product only, fad2 males (X⁺Y) and homozygous females (X⁺X⁺) had the 567-bp transgene products only, and the heterozygous fad2 females (X⁺X) had both bands together. X⁺ and X represent the X chromosome integrated with fad2 transgene and wild-type X chromosome, respectively.

Figure 4

PCR analysis of the 5' integration site. (A) Schematic diagram of the approximate positions of five primer sets used for PCR analysis of the 5' integration site. P1 (-54,088 to -53,884), P2 (-29,872 to -29,553), and P3 (-44,562 to -44,144) primer pairs were designed according to the sequence upstream of nucleotide 87,507,732 (+1), whereas the forward primers of P4 (from -46,680) and P5 (from -45,797) were designed corresponding to the X sequence. The reverse primer (5'-gccaagtgggcagttta-3') corresponded to the CMV enhancer sequence included in the transgene construct. X⁺ represents the transgenic X. The double-dashed line represents the foreign DNA molecules. (B) Gel analysis of the PCR products amplified from the fad2 or C57 mice. The 205-bp fragments using P1 primers were successfully amplified in X⁺X⁺, X⁺X, X⁺Y, or C57 samples. The 320-bp fragments using P2 primers and the 419-bp fragments using P3 primers were only amplified in the X⁺X and C57, not in X⁺X⁺ or X⁺Y, samples. The 1732-bp fragment amplified by P4 primers and the 799-bp fragment amplified using P5 primers were present in all fad2, but not C57, mouse samples, and they were sequenced directly. (C) Nucleotide sequence of the P5 primer pair (underlined) and the 5' integration region in fad2 mice. Sequences from the 5' initial nucleotide to +509 were determined by direct sequencing. The partial sequence from the 5' initial nucleotide to -1 was 100% identical to the region upstream of the 87,462,177th nucleotide of the XC1 region of the X chromosome. The sequence from -3 to +509 showed 100% identity to the transgene sequence. A 5'-TGT-3' sequence (-3 to -1) was shared by the transgene and the X chromosome as a very short homologous arm. Transgenic sequences were divided into two classes based on the presence of five additional nucleotides (TACTG). The sequence from -3 to +324 was 100% identical to the foreign complementary sequence (from 3580 to 3259). The sequence from +330 to the 3' end was 100% identical to the 5' initial sequence of the transgene.

Figure 5

Transgene mapping on the X chromosome. The map of mouse chromosome X shows that the entire 45,556-bp region from 87,462,177 to 87,507,732 of XC1 was replaced by the foreign DNA during the process of random integration.

Figure 6

Analysis of the sequences surrounding the junction sites. The junction sequences present in the fad2 mice are underlined. Identical nucleotides in fad2 and X chromosome are indicated (grey shading). Three or seven identical nucleotides (boxed with grey shading) existed in the AT-rich domain, presumably corresponding to the common sequence of the recombination of transgene and host genome.

Figure 7

RT-PCR analysis of the transgene and the X-linked sequences. Total RNA from liver, kidney, brain, muscle, and heart tissues of the C57 or homozygous fad2 females was analysed using the different sets of primers. (A) The X-linked Hprt gene (normalisation control) showed normal expression in all samples from the fad2 and C57 mice. (B) Transgene specific primers revealed that the fad2 gene was expressed only in the transgenic mouse, not in the C57 mouse. (C-F) Transcripts of the region surrounding the X insertion site (from -1123 of the 5' integration to +3069 of the 3' integration site) were not detected in any C57 tissue. In fad2 mice, the examined upstream sequences (-1123 to -977) did not amplify (C), whereas RT-PCR fragments (D-E) of the downstream regions from +859 to +2299 were amplified successfully. The distal downstream region (from +1952 to +3069) was not detected in any of the examined transgenic tissues (F). Amplification without reverse transcriptase in each samples showed no contamination.

Figure 8

Relative expression of the X-linked transgene. Sets of primers corresponding to fad2 or Gapdh were used for real-time RT-PCR analysis of the samples of transgenic or C57 kidney, brain, and liver. Expression data of fad2 were normalised to Gapdh expression and presented as the mean relative quantity (compared with X⁺X), with error bars representing the SEM. Student's t-test was used to calculate P values. Values with different superscripts were significantly different within same tissues (P < 0.05).

Figure 9

Random integration mechanism of foreign DNA by homologous illegitimate random integration and course of fad2 inserted into the X chromosome. (A) Identical foreign DNA injected into the nucleus of a eukaryotic cell is circularised and randomly cleaved by the endogenous restriction enzyme(s). It generates tandem concatemers by homologous recombination (steps 1-3, adapted from reference [2]). The concatemers are inserted into the host genome, mediated by homologous illegitimate random integration (HIRI), which depends on several identical nucleotide sequences (in red or blue) in the AT-rich domains on both sides, which serve as anchors for the two DNAs. Consequently, a host DNA region is replaced by the foreign DNA. During DNA replication, the repair mechanism of the cell induces foreign DNA integration into the host genome (steps 4-5). (B) Fad2 transgenic concatemers consist of a ≥322-bp complementary DNA and multiple copies inserted in a head-to-tail array by homologous recombination. During the HIRI process, 5'-TGT-3' and 5'-TTAATAG-3' (capital letters in blue) in the AT-rich domain, which exist in both foreign DNA and the X chromosome, mediate the illegitimate recombination of the transgene and the X chromosome as the homologous arms. When the homologous foreign DNA is inserted into the X chromosome, all of the 45,556 target nucleotides on the X chromosome are replaced by the foreign DNA.