Y. Makas. University of Baltimore.

With the use of Doppler echocardiography purchase genuine diovan on-line, it is possible to do this by detecting systolic regurgitation of the pulmonary valve buy discount diovan 80mg on line, which is caused by a jetting effect of the patent ductus arteriosus against the valve (see Fig buy diovan american express. Another technique is through the use of Doppler echocardiography during positive pressure ventilation order diovan without prescription, which transiently results in opening of the pulmonary valve and forward Doppler flow. Finally, an attempt to document coronary artery connections from the right ventricle should be made, recognizing that this can be difficult (see Fig. C: Subcostal imaging of the epicardial right coronary artery confirms dilation to suggest increased flow. D: Pulsed Doppler confirms systolic retrograde flow with diastolic antegrade flow in the coronary vessel with connections to the hypertensive right ventricle. Cardiac Catheterization If one is considering decompression of the right ventricle full hemodynamic and angiocardiographic investigations are required for the interventional management of the neonate with pulmonary atresia and intact ventricular septum. Although one can recognize echocardiographically the presence of large ventriculocoronary connections or fistulae, this technique does not allow the recognition of coronary artery stenosis or interruption, both of which have been documented in the newborn. Significant coronary arterial abnormalities with a right ventricle dependence for myocardial flow have been predicted by an evaluation of the size of the tricuspid valve relative to the patient size with a Z-score of less than −2. Note the tricuspid insufficiency (black arrow) and the atretic pulmonary valve (white arrow). The level of systemic arterial oxygen saturation data reflects the magnitude of pulmonary flow. Before the era of routine administration of prostaglandin, these infants became progressively hypoxemic coincident with functional and anatomic closure of the arterial duct. All these infants experience an obligatory right-to-left shunt at the atrial level. Unless there is pulmonary disease the pulmonary venous saturation is normal, or nearly so. The administration of prostaglandin, by virtue of its action on the arterial duct and the pulmonary vascular resistance, augments pulmonary flow and thus arterial saturation. This should be interpreted as consistent with lack of a proximal aortocoronary connection. The hemodynamic evaluation of the patient with the hypertensive right ventricle establishes right ventricular pressure at or above systemic levels. When dealing with the patient with massive cardiac enlargement, right ventricular pressure may be substantially less than systemic. These patients raise the possibility that the obstruction to pulmonary blood flow reflects functional rather than anatomic pulmonary atresia. In the presence of a nonrestrictive interatrial communication, the atrial mean pressures equalize, although there is often a small “a” wave gradient from the right to the left atrium. If true restriction to atrial flow is demonstrated it is important to consider a balloon atrial septostomy to avoid a low cardiac output syndrome, especially if decompression of the right ventricle is not to be included in the therapeutic tract. The right ventricle is at systemic levels of pressure or higher, and the end-diastolic pressure may be abnormally high, consistent with a noncompliant ventricular chamber. The finding of a subsystemic right ventricular pressure is consistent with a globally disadvantaged right ventricle. The right ventricle is usually thinned, and severe tricuspid regurgitation is often present. Angiocardiography remains an important imaging modality in patients with pulmonary atresia and intact ventricular septum (Figs. Although much of the intracardiac anatomy can be determined from a systematic approach P. Most importantly, right ventricular angiography will define the presence or absence of ventriculocoronary connections. Selective injection of the right ventricular outflow tract can differentiate severe stenosis of the pulmonary valve from membranous atresia (58). Selective left ventricular angiocardiography provides information about the form and function of the left ventricle and aortic valve, and this certainly complements echocardiographic imaging. Aortography in the neonate defines the laterality of the aortic arch, the caliber of the subclavian arteries, the site of insertion of the arterial duct, the caliber of the pulmonary arteries, and may exclude coarctation of the pulmonary artery at the insertion of the arterial duct that may preclude treatment with a stent to permanently maintain its patency or require a surgical patch at the time of placing a systemic-to-pulmonary artery shunt. In some patients, including newborns, selective coronary arteriography may be necessary to characterize fully the coronary arterial involvement. When right ventricular angiography does not demonstrate ventriculocoronary connections, one can be reasonably certain that coronary arterial stenosis or interruption or major fistulae with coronary–cameral flow will not be evident. In some patients with ventriculocoronary connections, a balloon catheter inflated in the right ventricle or catheter-induced tricuspid insufficiency with concurrent observation of the simultaneous electrocardiographic tracing may unmask a right ventricular–dependent coronary circulation. Extensive ventriculocoronary connections are demonstrated by frontal and lateral right ventriculograms. The anterior descending coronary artery demonstrates numerous caliber changes (arrows) consistent with arterial obstructions (see also Fig. A: The lateral projection demonstrates the presence of a communication between the right ventricle and the coronary artery. The left main coronary artery continues posteriorly as the circumflex vessel (Cx). B: In the frontal projection the aortic origin of the left coronary artery is obscured, however, a network of fine vessels within the leftward and anterior myocardium is delineated. Left ( C) and right ( D) axially angulated projections fail to demonstrate an anterior descending vessel. B: Retrograde selective injection of the left coronary artery in a long-axis oblique projection in the same patient clearly demonstrates the left ventricular myocardium perfused by the aortocoronary supply without significant coronary artery stenosis or occlusion. The angiocardiographic investigation of the patient with a hypertensive right ventricle requires right ventricular angiocardiography in frontal and lateral projections (see Figs. Further information can be obtained with angulated views of the right ventricular angiocardiogram in the setting of ventriculocoronary communications (see Fig. It remains to be seen whether advanced imaging techniques such as three- dimensional rotational angiography will augment the evaluation of coronary pathology in this condition (Fig. The reported surgical survival prior to the availability of prostaglandins was only 27% (59). Rarely an urgent balloon atrial septostomy may be required for a restrictive septum as pulmonary flow improves and should be considered in any neonate that demonstrates persistent low cardiac output with the usual therapeutic maneuvers. For the premature infant or the extremely small-for-gestational-age infant, a prolonged course of an E-type prostaglandin may be necessary before surgery is undertaken, although this is unusual in the current surgical era. Once pulmonary flow is established it is important to recognize that systemic oxygen saturation is related to the amount of flow into the pulmonary circulation. A very low pulmonary vascular resistance and conversely high systemic vascular resistance can result in low cardiac output despite a high saturation. The patient has an obligatory right-to-left shunt at atrial level hence a very high saturation usually reflects an enormous amount of pulmonary flow. The resulting low diastolic pressure and low cardiac output syndrome can result in compromised splanchnic and renal flow as well as ongoing acidosis and, in the extreme, cardiogenic shock. In addition this physiology can compromise oxygen delivery to the aortocoronary circulation resulting in myocardial ischemia. Surgical Surgical and other catheter-based interventional therapies have continued to change with an understanding of the heterogeneity of this disorder. The diagnostic and therapeutic algorithms are now quite diverse, depending on a wide number of morphologic variables (5,60,61,62,63,64). Among some centers, primary cardiac transplantation is also considered particularly when the coronary artery anatomy is complex. In evaluating an individual patient with this disorder, and assuming a neonatal presentation, different surgical strategies will be derived for the patient with massive cardiomegaly and a right-to-left ventricular pressure ratio below 1. Most infants with pulmonary atresia and intact ventricular septum will have some degree of right ventricular hypoplasia and hypertension; thus initial considerations should include the following: 1. Is the patient ultimately a candidate for a complete biventricular (including the so-called one-and-a-half ventricle) repair or a univentricular total cavopulmonary (Fontan) palliation? Is there a main pulmonary trunk in continuity with the imperforate pulmonary valve? Ideally, one would like to achieve a biventricular repair and the initial approach should allow right ventricular decompression by some form of right ventricular outflow tract reconstruction, whether surgical pulmonary valvotomy, outflow tract patch, or catheter-based perforation and dilation of the pulmonary valve. Success may be predicted by analysis of the relative size of the right and left ventricles in the fetus (65). If there are ventriculocoronary connections and the majority or the entirety of the coronary circulation is right ventricular dependent, clearly the patient should be placed on a univentricular palliation algorithm (66,67,68). However it is important to recognize that right ventricular myocardial hypertrophy, myocardial fiber disarray, and endocardial fibroelastosis may contribute to right ventricular restrictive physiology that can affect exercise tolerance and functional outcome even in the setting of a biventricular repair (69). This notion of restrictive physiology challenges the wisdom of pursuing a biventricular repair in the setting of a “borderline” right ventricle wherein the patient may be functionally better served by a univentricular strategy (70).

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Biventricular hypertrophy may be seen particularly in cases of left ventricular outflow tract obstruction or significant left ventricular volume overload generic diovan 40 mg on line. Note the narrow mediastinum due to the direct anterior–posterior relationship of the aorta and pulmonary artery 160mg diovan amex. In cases where the aorta and pulmonary trunk exhibit a direct (or somewhat direct) anterior–posterior relationship order diovan australia, the mediastinal shadow is narrow and not as prominent as usual discount 160 mg diovan fast delivery, leading to the classic “egg on a string appearance” of the cardiac silhouette (Fig. Pulmonary vascular markings correlate inversely to the severity of left ventricular outflow tract narrowing in patients with transposition of the great arteries, ventricular septal defect and left ventricular outflow tract obstruction. Echocardiography In the current era, the diagnosis is confirmed by transthoracic echocardiography. Two-dimensional (2-D) echocardiography in the parasternal long-axis view demonstrates the pulmonary artery originating from the left ventricle coursing posteriorly (and bifurcating into branch pulmonary arteries) and the aorta originating from the right ventricle in the same plane coursing anteriorly (Fig. Doppler interrogation shows flow into the posterior vessel (pulmonary artery) via the arterial duct. The origin of the pulmonary trunk from the left ventricle and the aorta from the right ventricle is also seen in other views, for example, in sweeps in the subcostal and parasternal apical chamber views. The parasternal short-axis view is helpful in determining the exact relationships of the aorta (identified as the great vessel from which the coronary arteries are seen originating) and pulmonary artery (identified as the great vessel that bifurcates) (Fig. A number of pertinent anatomic details should be obtained from transthoracic echocardiography that are relevant to the medical, interventional catheterization and surgical management of these patients. The size of the patent ductus arteriosus and direction of flow is important for medical management, prior to and while the patient is receiving prostaglandin infusion. Atrial level shunts, for example, a patent foramen ovale or secundum atrial septal defect, should be delineated, particularly with reference to overall anatomic adequacy of the size of the shunt/shunts and velocity of flow. Juxtaposition of the atrial appendages can be seen in transposition of the great arteries (37), with left-sided juxtaposition of the right atrial appendage being more common than right-sided juxtaposition of the left atrial appendage. The orifice of the juxtaposed right atrial appendage can be mistaken for large atrial septal defect (which, in a profoundly P. This may have important bearing on an eventual balloon atrial septostomy (detailed in “Balloon Atrial Septostomy” section). A number of important anatomic and presurgical details should be determined, including the relative size of the semilunar valve annuli and detection of abnormal semilunar valve morphology. The origins of the coronary arteries and their branching (and presence or absence of an intramural course) is an important finding that is accurately diagnosed by echocardiography (Fig. In patients with transposition of the great arteries, ventricular septal defect and left ventricular outflow tract obstruction, the precise mechanism of obstruction (e. The pulmonary artery is identified as the vessel bifurcating into branches, for example, the right and left pulmonary arteries. A: The left coronary artery is seen by 2-D echocardiography (arrow) with flow seen by color flow Doppler (B, arrow). C: The right coronary artery is seen by 2-D echocardiography (arrow), with flow seen by color flow Doppler (D, arrow). Note the pulmonary artery (left arrow) draped over the aorta (Lecompte maneuver) and the anterior and leftward located translocated left coronary artery (right arrow). This is advantageous in preparing and counseling families of the implications of the disease and its prognosis. Additionally, delivery at, or transport to a facility equipped to handle these newborns can be planned, along with appropriate postnatal management. Cardiac Catheterization/Angiography These days, cardiac catheterization is not needed for diagnosis. In a previous era, the “laid-back” angiogram was performed to evaluate the coronary arteries (45). This can also be performed if the patient undergoes a preoperative catheterization for other purposes, or the coronary artery pattern cannot be readily discerned by echocardiography (although rare), and the information is felt mandatory by the surgical team (not usually necessary). This is performed by angulating the frontal camera in an extreme caudal projection. A balloon occlusion angiogram in the ascending aorta with a catheter that has side holes proximal to the balloon, for example, a Berman angiographic catheter (Arrow International, Inc. This same angulation may be used from the femoral arterial approach after an arterial switch operation to examine the coronary arteries if needed. Selective coronary angiography can be safely performed even in very small infants. On the right, a dilated neo-aortic root (arrow) is seen, a common long-term finding in these patients. Extreme caudal angulation is placed on the anterior–posterior projection (∼45 degrees) and a balloon occlusion angiogram is performed in the aorta. Note that this newborn has a single coronary artery ostium (arrow), giving rise to the right coronary artery (single asterisk), left anterior descending coronary artery (double asterisks) and circumflex coronary artery (triple asterisks). Natural History and Management Natural History and Immediate Outcomes Untreated, transposition of the great arteries is a fatal disease. A unique study from a large database between 1957 and 1964 in the state of California performed by Liebman et al. The advent of the Blalock Hanlon procedure (surgical atrial septal defect creation) (47) and various partial venous redirection procedures (e. In addition to these procedures, the less invasive balloon atrial septostomy procedure (2) dramatically changed the natural history and early survival of these infants (50,51). A: The operation is performed utilizing hypothermic cardiopulmonary bypass with cannulation of the superior and inferior vena cavae. The aorta is cross-clamped and the myocardium is protected with intermittent doses of cold cardioplegic solution. B: An atrial septal flap is created by incising the septum on its anterior, inferior, and superior aspects. Note that there is atrioventricular concordance so that the mitral valve is left sided and the tricuspid valve right sided. C: The flap is now turned down into the left atrium and is sutured around the anterior, inferior, and superior margins of the orifices of the pulmonary veins (blue suture line). This flap isolates the pulmonary veins from the left atrium and forms the roof of the pulmonary venous chamber and the floor of the systemic venous chamber. The posterior right atrial wall is then used as a flap to construct the anterior aspect of the vena caval pathway to the mitral valve. It is sutured around the orifices of the superior and inferior vena cavae and brought to the edge of the septum between the tricuspid and mitral valves (red dotted line). The anterior right atrial wall is used as a flap to construct the pulmonary venous pathway from the left atriotomy to the tricuspid valve (orange dotted line). Pulmonary venous return is directed to the aorta via the tricuspid valve and right ventricle (red arrow) and systemic venous return is directed to the pulmonary artery via the mitral valve and left ventricle (blue arrows) leading to a physiologic correction at the atrial level. This leaves the morphologic right ventricle as the systemic ventricle and the tricuspid valve as the systemic atrioventricular valve. Results of the Senning and Mustard procedures were reproducible with early survival approaching generally 90% in experienced centers and patients achieved a normal oxygen saturation. Data from the Congenital Heart Surgeons Study showed the early mortality from the Senning and Mustard procedures to be 11% and 0%, respectively (54). Further follow-up showed survival for these atrial redirection procedures was 90% and 85% at 1 month and 5 years, respectively, with survival in the Mustard group being better (55). A: The operation is performed utilizing hypothermic cardiopulmonary bypass with cannulation of the superior and inferior vena cavae. The aorta is cross-clamped and the myocardium is protected with intermittent doses of cold cardioplegic solution. B: The atrial septum and majority of the limbus are resected to create a large atrial septal defect that extends to the superior and inferior vena cava. C: The large interatrial communication has been created exposing the pulmonary veins. Note that there is atrioventricular concordance so that the mitral valve is left sided and the tricuspid valve right sided. D: An intra-atrial baffle (shaded), usually of pericardium, is constructed to direct the vena caval flow to the mitral valve.

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Peptic strictures are characteristically short and have smooth outlines with tapering ends (Fig order diovan 160 mg without prescription. Dilatation of the oesophagus There are two main types of oesophageal dilatation – Achalasia obstructive and non-obstructive: Achalasia is a neuromuscular abnormality resulting in • Dilatation due to obstruction is associated with a visible failure of relaxation at the cardiac sphincter effective diovan 160mg, which presents stricture generic 40 mg diovan free shipping. The patient with a carcinoma usually presents at barium swallow examination as a smooth order diovan cheap, tapered nar- with dysphagia before the oesophagus becomes very dilat- rowing, always at the lower end of the oesophagus (Fig. There is a short smooth stricture at the oesophagogastric junction with an ulcer crater within the stricture (arrow). A web may be an isolated fnding, but The disease involves the oesophageal muscle, resulting in the combination of a web, dysphagia and iron defciency dilatation of the oesophagus, which resembles an inert tube anaemia is known as Plummer–Vinson syndrome. A pharyn- An oesophageal web is a thin, shelf-like projection arising geal pouch arises through a congenital weakness in the from the anterior wall of the cervical portion of the oesopha- inferior constrictor muscle of the pharynx and comes to lie Gastrointestinal Tract 151 Fig. The very dilated oesophagus containing food residues shows a smooth narrowing at its lower end. It may reach a very large size and can cause displacement and compres- sion of the oesophagus. In oesophageal atresia, the oesophagus ends as a blind pouch in the upper mediastinum. A plain abdominal flm will show air in the bowel contrast agents is potentially dangerous because the con- if a fstula is present between the tracheobronchial tree and trast may cause respiratory problems if it spills over into the oesophagus distal to the atretic segment. There is a shelf-like indentation (arrow) from the anterior wall of the upper oesophagus. There is a localized indentation caused by the anomalous artery as it passes behind the oesophagus (arrow). The frst two types also have an oesophagotracheal fstula distal to the atretic segment and will show air in the stomach. The stomach is distended with a gas-producing agent, and an intravenous injection of a short-acting smooth muscle relaxant is often given. Films are taken in various positions with the patient both erect and lying fat, so that each part of the stomach and duodenum is shown distended by barium and also distended with air but coated with barium to show the mucosal pattern (Fig. The duodenal cap or bulb arises just beyond the short pyloric canal, and the duodenum forms a loop around the head of the pancreas to reach the duodenojejunal fexure. Diverticula arising beyond the frst part of duodenum are a common fnding and are usually without clinical signif- cance (Fig. The pouch which are more rapidly resorbed than barium and impor- lies behind the oesophagus, which is displaced forward. The body and the antrum of the stomach together with the duodenal cap and loop are coated with barium and distended with gas. Note how the fourth part of the duodenum and duodenojejunal fexure are superimposed on the body of the stomach. If the • Assessment of functional patency/gastric emptying follow- stomach is not distended during the scan, any thickening ing gastroenterostomy or anti-obesity surgery of the gastric wall could be misinterpreted as being a mass. It enables the mucosa of the stomach and about 100 mL of tap water to drink (acting as a negative duodenum to be directly inspected and biopsied. The main contrast) as well as a smooth muscle relaxant, in order to indications for its use are summarized in Box 6. The stomach has been distended by oral water contrast and the use of an intravenous smooth muscle relaxant. With duodenal ulceration, the duodenal cap (bulb) may be very deformed by scarring. Gastric carcinoma Emphasis is placed on the diagnosis of early gastric cancer, which is confned to the mucosa, as early treatment has a much better prognosis. In the past, high quality barium meal examination has been used as a screening tool in some countries to detect early gastric carcinoma. At barium examination, gastric carcinoma typically pro- duces an irregular flling defect with alteration of the normal mucosal pattern (Fig. There are a number the preoperative staging of patients with gastric cancer as of large flling defects in the antrum and body of the stomach. Several lymph nodes (short black arrows) and a liver metastasis (long black arrow) are also seen. There is a smooth ovoid mass arising from the anterior wall of the stomach (arrow). The tiny gastrinoma is seen as a brightly enhancing lesion in the wall of the duodenum (arrow), on the arterial phase of enhancement. Gastrinomas usually secrete gastrin, resulting in of the stomach resulting in a smooth, round, submucosal increased gastric acidity and peptic ulceration. Gastrino- flling defect, which may ulcerate as the tumour enlarges mas are often very small and typically enhance brightly in (Fig. Gastric polyps A leiomyoma is a submucosal tumour which, as well as projecting into the lumen of the stomach, may have a large Gastric polyps may be single or multiple. Even with high quality radio- Neuroendocrine tumours of the stomach and duodenum graphs it is often impossible to distinguish benign from include gastric carcinoid tumours and gastrinomas. Other intraluminal defects within the stomach incidentally in the stomach often as multiple small (<1 cm) include food or blood following a haematemesis. Sometimes tumours on a background of pernicious anaemia/chronic ingested fbrous material, such as hair, may intertwine atrophic gastritis. Lymphadenopathy surrounds the inferior vena antrum is also distended by the ingested material (black arrow). The appearance of primary gastric may or may not be possible to identify an actual ulcer crater lymphoma is typically of an extensive area of diffuse thick- • Carcinoma of the antrum • Duodenal, ampullary and pancreatic carcinoma ening of the gastric wall (Fig. There may be exten- • Acute or chronic pancreatitis, including pseudocyst sive, bulky lymphadenopathy adjacent to the tumour. Prolonged delay in a patient mediastinum through the oesophageal hiatus in the dia- with a dilated stomach containing food residues needs to phragm. In adults, the causes of gastric outlet obstruc- hernia exist: sliding and rolling. In infants, pyloric stenosis is by far the commonest cause The commoner type is the sliding hiatus hernia, where of gastric outlet obstruction. Often, the diagnosis is made the gastro-oesophageal junction and a portion of the clinically and can be confrmed with ultrasound, which has stomach are situated above the diaphragm (Fig. Ultrasound shows a thickened, cardiac sphincter is usually incompetent, so refux from elongated pyloric canal (Fig. A small A large hernia, particularly one of the para-oesophageal sliding hernia may be demonstrated in most people during type, may not be reduced when the patient is in the erect a barium meal examination, provided that enough manoeu- position. It is, therefore, diffcult to assess the signifcance of a small hernia with little or no refux. The fundus of the stomach and the gastro-oesophageal junction (arrow) have herniated through the oesophageal hiatus and lie above the diaphragm (dotted line). A endoscopy is diffcult to perform due to the long and tortu- ous nature of the small intestine. Capsule endoscopy, which requires the patient to ingest a tiny capsule containing a camera, may be helpful in some cases (Fig. Gastrointestinal Tract 161 • Computed tomography has a role in the assessment of small bowel disease as it can show thickening of the bowel wall, an important sign of infammatory bowel disease and lymphoma, and occasionally mucosal irregularity and small bowel neoplasms. The patient fasts for 4–6 hours prior to the procedure and then may be given oral Gastrografn or water to delineate the small bowel. Intravenous iodinated contrast medium is injected to demonstrate vessels and enhancing structures. In some cases, it can demonstrate small bowel thickening and free fuid associated with infammatory conditions, and can demonstrate a thickened appendix suggestive of appendicitis and occasionally the presence of an intussusception. A nasogastric tube is also present, projected over the left • Nuclear medicine techniques, using meta- upper quadrant. Technetium-99m (99mTc) may be used to detect Meckel’s Imaging techniques diverticulum. Standard imaging techniques and their indications are: • Small bowel follow-through (small bowel meal) (Fig. Normal appearances of the small bowel The patient drinks about 200–300 mL of barium and its passage through the small intestine is observed by taking The normal small intestine (see Fig. This can be a time-consuming procedure and usually takes The terminal portion of the ileum enters the medial aspect 2–3 hours, but the transit time is very variable. As the terminal tions for a barium examination of the small bowel are listed ileum may be the frst site of disease, this region is often in Box 6.

The diploid nature of higher organisms means that mutants that fall into this class may be analysed in their heterozygous (+/−) state buy diovan 80 mg with visa. Knock-outs that fall into the last category (no observable phenotype) may arise as a result of genes acting in parallel pathways compensating for each others’ functions buy diovan with paypal. It is also possible that the techniques are simply too crude to detect any subtle differ- ences between the wild-type and the knock-out animals cheap generic diovan canada. The complexity of animal genomes also means that a knock-out may have a profound effect in one strain of mouse order 160mg diovan mastercard, but quite a different effect in another. Ideally, knockout experiments should be performed in a variety of strain backgrounds, but the length of time required to do that, and the costs involved, often preclude this analysis. One problem with this type of approach for producing transgenic ani- mals, which we have seem previously when looking at engineering in plants (Chapter 11), is that the selectable maker gene is transferred to the transgenic animal. The high-level expression of an antibiotic-resistance gene within a transgenic animal is generally undesirable. The expression of the marker may induce the abnormal expression of other neighbouring genes, and the potential for transfer of the marker gene to non-transgenic animals should be avoided. There are many instances where the expression of an inserted transgene is required only in a specific tissue or set of cells. This can readily be achieved by constructing the foreign gene such that it is under the control of a tissue- specific promoter. Such an approach works well, provided that a suitable tissue-specific promoter is available (Table 13. Conditional knock-outs can also be produced, again using the loxP-Cre site-specific recombination system (Gossen and Bujard, 2002). If, for example, the knock-out of a gene results in an embryonic-lethal phenotype, then it may be necessary to delete the gene from the genome after the animal has been born. Adapted from Lewandoski (2001) Promoter Gene normally Tissue or cells Reference controlled of expression Alb Albumin Liver (Postic et al. In addition, the transgenic animal is also modified to carry a copy of the gene encoding the Cre recombinase under the control of an inducible promoter, e. Mx1 is part of the mouse viral defence system and is transcriptionally inert in healthy mice (Hug et al. Rather than constructing a transgenic mouse containing both the tissue- specific promoter expressing the Cre recombinase and the target gene sur- rounded by loxP sites, a series of transgenic mice have been constructed that each contain a different tissue-specific promoter controlling the expressing of Cre. These can then be used as a ‘bank’ of mice strains to which transgenic mice containing a particular floxed gene can be crossed. Mating these strains will result in the formation of progeny in which the gene in inactivated only in those tissues that express Cre (Gu et al. This means that a single transgenic floxed gene can be deleted in a variety of tissues without having to resort to further in vitro manipulation. The tetracycline-inducible expression system (see Chapter 8) may be used to drive Cre expression to regulate knock-out function. In the absence of tetracycline, the Cre gene is expressed and will induce site-specific recombination between two loxP sites. In the presence of tetracycline, the Cre gene will not be expressed and recombination will not occur (St-Onge, Furth and Gruss, 1996). If the nucleus of a differentiated cell is introduced into an enucleated egg then, under appropriate conditions, the nucleus can become ‘reprogrammed’ such that development of the animal reoccurs. The production of cloned animals – all of which have orig- inated from a single, possibly recombinant, cell line – has several potential uses. We have discussed previously that the expression level of recombinant protein production is not strictly inherited (Chapter 12). Therefore, the ability to create large number of animals each expressing identical levels of, say, a therapeutic protein can only be achieved using cloned animals. Over 50 years ago it was discovered that the nuclei of blastocyst frog cells could be implanted into eggs that lacked a nucleus to created a series of cloned frogs that were identical to the donor cells (Briggs and King, 1952). It was found, however, that as the donor cells became more differentiated, it became increasing difficult to reprogramme them to produce new animals. The few embryos cloned from differentiated cells that survived to become tadpoles grew abnormally. This led to the speculation that genetic potential diminished as a cell differentiated and that it was impossible to clone an organism from adult differentiated cells. In 1975, however, John Gurdon developed a method of nuclear transfer using fully differentiated cells and Xenopus eggs (Gurdon, Laskey and Reeves, 1975). Delicate needles and a powerful microscope were used to suck the nucleus from a frog oocyte to produce an enucleated oocyte. With the genetic material removed the enucleated oocyte would not divide or differentiate even when fertilized. Using the same equipment, the nuclei of keratinized skin cells of adult Xenopus foot-webs were transfered into the enucleated oocytes. Many of these new cells behaved like normal fertilized eggs and were capable of producing tadpoles. Since the tadpoles arose from the cells of the same adult, they all contained the same genetic material and were clones of each other produced from apparently fully differentiated cells. A somewhat modified procedure has been used recently to produce cloned mammals (Figure 13. This was first achieved by taking cells from the blastocyst stage of a sheep embryo and fusing them with enucleated eggs (Smith and Wilmut, 1989). The reconstituted cells were subjected to a brief electrical pulse to stimulate embryonic development prior to implantation into a surrogate ewe. The cells of an adult sheep (sheep 1) are fused with the enucleated eggs of a sheep of a different breed (sheep 2). The fusion between the two is grown in culture to the blastocyst stage prior to implantation into a surrogate ewe. The resulting lamb contains the nuclear genome of sheep 1 nuclei of cultured embryonic cells (Campbell et al. This last example produced probably the most famous sheep in the world – Dolly (Box 13. The success of these experiments appears to be dependent on the synchronization of the cell cycles of the donor and recipient cells that are to be fused. In the case of Dolly, quiescence of the donor cell was induced prior to the cell fusion process. Mammary gland tissue of a 6-year-old Finn Dorset ewe was used to prepare a primary cell culture. This culture contained a mixture of mammary epithelial cells (>90 per cent), myoepithelial cells and fibroblasts. An important step in the success of the cloning process was to induce these donor cells to exit their growth cycle and enter the G0 phase of the cell cycle before nuclear transfer. After 6 days of culture, 29 of the 277 reconstructed embryos had developed into a morula or blastocyst. One, two or three embryos were transferred to Scottish Blackface ewes and allowed to develop to term. The 29 morula/blastocysts were transferred to 13 differ- ent ewes, and from these only one became pregnant. On July 5 1996 after 148 days pregnancy, the normal duration for her breed, Dolly – a Finn Dorset sheep – was born with a healthy birth weight of 6. Dolly, a sheep derived from a mammary gland cell, was named after the singer Dolly Parton. Further analysis indicated that she was indeed derived from the cells of the mammary gland of the donor sheep, rather than from a contaminating cell (Ashworth et al. She is not, however, an exact clone of the sheep whose cells were used to create her. It also is interesting to note that the scientific paper in which Dolly was introduced to the world (Wilmut et al. Perhaps the authors realized the potential impact of their findings and chose less inflammatory language to describe their results. Dolly subsequently grew into an adult sheep have bore her own offspring (Box Figure 13. Finn Dorset sheep have an average life expectancy of about 12 years, but in January 2002 Dolly was reported to be suffering from arthritis, which is highly unusual for a sheep of her age.

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