Transplantation of a female aorta to into a male recipient enabled us to analyze the newly formed tissues, and especially cartilage, using the polymerase chain reaction (PCR) technique for detection of the SRY gene (attesting to the presence of the Y chromosome). Tracheal cartilage biopsy samples were obtained from male and female control minipigs. Biopsy samples were also obtained from the graft in areas grossly determined to have nests of cartilage in two male animals that survived 10 months and 11 months, respectively, after transplantation of a female AA. Cartilaginous rings from control minipigs and tissue from the graft (Fig 1) were dissected (0.5 cm) for DNA extraction and molecular analysis. Tracheal samples were incubated overnight at 56°C with proteinase K, and total DNA was extracted by classical phenol/chloroform technique, as previously described.
SRY gene amplification was carried out on each sample (male trachea, female trachea, graft). As a tracer for DNA extraction and amplification steps, we used primers for the IGF1 gene as a positive control (Table 1). Amplification was carried out in a DNA thermal cycler (Perkin-Elmer Applied Biosystems; Foster City, CA). PCR was set up in a final volume of 50 |j,L, with 29 pmol of each primer (specific of the Sus scrofa species), 10 X Taq polymerase buffer, 0.75 mmol/L of magnesium chloride, 200 |j,mo]/L deoxynucleoside triphosphates, 2.5 U of Taq polymerase, and 5 |j,L of extracted DNA (ie, 50 ng/^L). The PCR products were 133 base-pair for SRY and 116 base-pair for IGF1. Thermal cycling was as follows: denaturation at 95°C for 10 min, 40 cycles of 94°C for 30 s; 58°C for 30 s; 72°C for 20 s, and final incubation at 72°C for 5 min. In case of negative results for SRY gene amplification, the presence of correct amplification of the IGF1 gene was used to control false-negative SRY gene amplification results. Fourteen pigs underwent replacement of the cervical trachea, 7 via simple cervicotomy and 7 with an associated partial sternotomy. Thoracic trachea replacement was attempted in seven pigs. Cervical trachea replacement was straightforward from a surgical perspective. In contrast, thoracic tracheal replacement was much more challenging, particularly during the distal anastomosis step. Indeed, despite preoxygenation, the pigs did not tolerate apnoea for > 1 min. The first two attempts resulted in peroperative hypoxemic death, and led us to use of the right lung “for ventilation” in the next two pigs. Improvement in the ventilation strategy allowed the replacement of the entire intrathoracic trachea and to splinting the aortic graft with a Y -shaped stent in the last three pigs.
Figure 1. Trachea at 50 weeks: extraluminal view (top) and intraluminal view (bottom). Graft is delineated by large arrows. Cartilaginous formations are shown by small arrows. The location of the patch biopsy (°) for SRY gene detection is indicated, as is the posterior membrane (*). CB = cranial bronchus; RMB = right main bronchus; LMB = left main bronchus.
Table 1—Characteristics and Sequences of Primers and Probes Used in the PCR Assay
|Gene Target||Primer Sequences|
|Sus scrofa SRY||5′ -GACAATCATAGCTCAAACGATG-3′|
|Sus scrofa IGF1||5′-GCAGCTGAAGCGCCTGGAGAAC-3′|