Use of single stranded targeting DNA or negative selection does not further increase the efficiency of a GGTA1 promoter trap

Abstract

Although several techniques have been developed to create gene knockouts in pigs, homologous recombination will continue to be required for site-specific genome modifications that are more sophisticated than gene disruption (base changes, domain exchanges, conditional knockouts). The objective of the present paper was to improve the efficiency of homologous recombination in porcine fetal fibroblasts, which would be used to produce gene knockout pigs by somatic cell nuclear transfer. A promoter-trap was used to enable selection of GGTA1 targeted cells. Cells were transfected with either a single stranded or double stranded targeting vector, or a vector, with or without a negative selectable marker gene (diphtheria toxin-A). Although targeting efficiencies were numerically lower for single stranded targeting vectors, statistical differences could not be detected. Similarly, the use of a negative selectable marker (in cis or trans) provided numerically lower targeting efficiencies, statistical differences again could not be detected. Overall, the targeting efficiencies ranged from 1.5×10-5 to 2.5×10-6 targeting events per transfected cell. Given the results, it may be applicable to investigate multiple enrichment techniques for homologous recombination, given that every targeted locus is different.

Keywords: Gene targeting; Homologous recombination; Promoter-trap; Singlestranded DNA; Swine; Transfection efficiency; Transgenic.

Figure 1

Schematic of the test plasmids. A, B, C and E are graphical representations of each targeting construct. Each construct included the corresponding porcine GGTA1 genomic sequences, used for the 5` and 3` recombination arms in the knockout vectors. Each construct included a mammalian optimized neomycin resistance cassette utilizing an IRES (internal ribosome entry site), which functions as a translation initial site for the neomycin protein, and a simian virus 40 (SV40) poly (A). Each construct also contains a human decay accelerating factor (hCD55) cassette, driven by the CAG promoter (cytomegalovirus early enhancer element and chicken beta-actin promoter). Constructs A, B, and C all contain a λ attB site located between the hCD55 cassette, and the SV40 poly (A) represented by a solid vertical bar. Construct E has a φC31 attB site located at the same location, represented by an open triangle. Constructs B and C contain one and two truncated diphtheria toxin- (tDT) genes, respectively. Construct D is a representation of the pDT-α vector used as a co-transfected plasmid. pDT-α contains the coding sequence for diphtheria toxin-alpha cassette driven by the PolII promoter, and contains a SV40 poly(A).

Figure 2

Graphical representation of potential plasmid integration sites. The two integration possibilities using this type of vector yield either a site-specific or random integration. The use of promoter trap in the vector requires that integration occur near an endogenous promoter, for proper transcription of the neomycin resistance gene. This allows for both integration possibilities, but with the use of the truncated diphtheria toxin- (tDT), as a negative selectable marker, the random integration possibilities should be reduced. Short arrows, along with their respective names, indicate the names and positions of the primers used for upstream and downstream PCR assays. For the upstream PCR assay, the primers were GLR_L1 and GSL_R2 for the forward and reverse primers, respectively. For the downstream PCR assay, the primers were GSR_L1 and GLR_R1 for the forward and reverse primers, respectively. The short bars indicate the location of the probe used for Southern blot analysis. The predicted size of Southern hybridization bands with HindIII digestion, for both the endogenous GGTA1 locus and the GGTA1 targeted locus, is as indicated.

Figure 3

Representative PCR screening from colony screening efforts. The result of a single PCR product for each assay was the same for screening colonies, fetuses, and piglets. The upstream assay was generated using GLR_L1 as the forward primer and GSL_R2 as the reverse primer. The resulting 5.5 kb PCR product can only be generated in the event of a targeting event, since the GLR_L1 primer is flanking the 5` targeting arm, and the GSL_R2 primer is located within the IRES sequence. In this figure, samples 4, 5, 12, 16, 19, 20, 21, and 24 are confirmed targeted by the upstream PCR. The downstream assay was used as a confirming diagnostic on upstream assay positive colonies. The downstream PCR assay was generated using GSR_L1 as the forward primer, and GLR_R1 as the reverse primer. The resulting 3.8 kb PCR product can only be generated when targeted, since the GSR_L1 primer is located within the hCD55 sequence, and the GLR_R1 primer is flanking the 3` targeting arm. Samples 4, 5, 12, 16, 19, 20, 21, 24, and 25 (not shown in Upstream PCR Assay) are confirmed targeted by both PCR assays. Sample 9 does not have a PCR product in the downstream PCR assay, and denoted as a random integrant. The controls used in the assays; negative controls: H20 used as template and wild-type genomic DNA; positive controls: previously targeted colonies. The standard was the λ genome digested with BstEII.

Figure 4

GGTA1 +/− knockout piglets at 3 days of age. Piglets were cloned from colony clone B.1.8, allele targeted with pBB7, and born on December 13, 2010.

Figure 5

PCR screening of GGTA1 +/− fetuses and piglets. The downstream (3.8 kb PCR product) and upstream (5.5 kb PCR product) PCR assays, are as previously described. The standard is the λ genome digested with BstEII. The samples represented are: (1) empty lane, (2) fetus 0903-1, (3) fetus 0903-2, (4) fetus 0903-3, (5) fetus 0903-4, (6) fetus 0903-5, (7) fetus 0903-6, (8) fetus 0903-7, (9) fetus 0903-8, (10) piglet 177-1, (11) piglet 177-2, (12) piglet 177-3, (13) piglet 177-4, (14) piglet 177-5, (15) piglet 177-6, (16) piglet 177-7, (17) piglet 177-8, (−) control sample H2O, (−) control wild type cell line genomic DNA. The samples are the same for both PCR assays.

Figure 6

Southern blot analysis of GGTA1 +/−. Representative analysis of DNA from day 35 fetus #0903-2. Lanes 1 to 3 are HindIII-digested genomic DNA from 1: wild type cell line (104821), 2: GGTA1 +/− fetus (#0903-2), and 3: wild type cell line (104821), with the equivalent of 2 molecules per porcine genome of a control plasmid, which contained probe sequences. A 691 bp and 657 bp PCR DNA fragment isolated from genomic DNA served as the probes. The 11.2 kb band represents the endogenous GGTA1 gene, the 8.6 kb band represents the disrupted GGTA1 locus, and the 7.2 sskb band represents 2 molecules of the probe control vector. All lanes were from the same Southern blot. To remove unrelated data lane, 3 was moved next to lanes 1 and 2.